Pharmaceutical compositions and methods for countering chemotherapy induced cardiotoxicity

ABSTRACT

This disclosure provides methods and pharmaceutical compositions for reducing or eliminating cardiotoxicity, particularly cardiotoxicity induced by a cancer treatment or other therapy. In some cases, the methods and compositions prevent or reduce cardiotoxicity caused by anthracycline treatment. The methods provided herein often comprise administering a protective agent such as myricetin, tricetin, robinetin, ficetin, vitexin, quercetin, dihydrorobinetin, kaempferol, 7,3′,4′,5′-tetrahydroxyflavone, and myricitrin in conjunction with the administration of a cancer drug or other treatment. They may comprise administering a protective agent in combination with dexrazoxane. The compositions provided herein include co-formulations of a protective agent with a different protective agent or with a cancer treatment (e.g., anthracycline drug).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/064,576, filed Oct. 6, 2020, now issued as U.S. Pat. No. 11,166,939,which is a continuation of U.S. patent application Ser. No. 16/737,849,filed Jan. 8, 2020, now issued as U.S. Pat. No. 10,874,633, which is acontinuation of U.S. patent application Ser. No. 16/075,569, filed Aug.3, 2018 and adopts International filing date of Feb. 3, 2017, now issuedas U.S. Pat. No. 10,806,716, which is a National Stage application under35 U.S.C. § 371 of International Application No. PCT/US2017/016582,filed Feb. 3, 2017, which claims the priority benefits of U.S.provisional application Ser. No. 62/291,480, filed Feb. 4, 2016, andU.S. provisional application Ser. No. 62/348,102, filed Jun. 9, 2016,the disclosure of each of which is incorporated herein by reference intheir entireties.

BACKGROUND

Cardiotoxicity and congestive heart failure are serious side-effects ofoncological therapies, most prominently those comprising anthracyclines,which are administered to greater than one million cancer patients peryear and half of all childhood cancer patients. Adverse cardiac sideeffects are also observed in patients treated with protein kinaseinhibitors and antibody-based biologics that target protein kinase.Certain reductions in heart failure rates have been achieved by cappingthe maximal doses of anthracyclines and by changing their administrationschedules, all of which severely limits the therapeutic potentials ofthese anticancer agents. The cardiotoxicity of cancer drugs can alsopreclude those patients with pre-existing cardiac conditions fromreceiving treatment.

Anthracyclines are generally a class of compounds that have thestructural core of anthracene. They often are highly effectivechemotherapeutics and therefore are used for the treatment of manycancers, including leukemias, lymphomas, breast, uterine, ovarian,bladder cancer, and lung cancers and are often used in childhood cancertreatment regimens. Some anthracycline drugs include doxorubicin,daunorubicin, idarubicin, and epirubicin. Although the exact mechanismsmay yet to be validated, anthracyclines have been reported to work byinhibiting DNA and RNA synthesis, promoting free radical formationthrough redox cycling, with iron promoting the conversion of superoxideinto hydroxyl radicals; inhibiting topoisomerases (e.g., topoisomerasesIIα and/or IIβ); and evicting histones from open chromosomal areas.

A common side effect of anthracycline use is associated withcardiotoxicity, which is dose dependent and may also result fromcumulative exposures. Cardiotoxicity, in some instances, may result fromthe formation of toxic reactive oxygen species through redox cyclingduring the metabolism of anthracyclines and from the formation ofdouble-stranded DNA breaks caused by inhibition of topoisomerase II. Thereactive oxygen species (ROS) may activate apoptotic pathways, leadingto cell death in both cancer and normal cells. Cardiomyocytes may besensitive to the oxidative stress. Cardiac mitochondria can be easilyinjured by anthracycline and anthracycline-iron complexes, which have ahigh affinity for dianionic phospholipid cardiolipin that is present ata high concentration in the inner mitochondrial membrane.

Some protein kinase inhibitors-including small molecule and biologicinhibitors may also cause cardiotoxicity. Protein kinase inhibitors area wide class of compounds that inhibits the activity of protein kinasesand can be used in cancer treatments. Tyrosine kinases regulate avariety of cellular functions including cell growth (e.g., epidermalgrowth factor (“EGFR”) and dysregulation may lead to certain forms ofcancer. Inhibition of such tyrosine protein kinases may be accomplishedby using small molecules that bind to the ATP pocket of a given proteinkinase, blocking it from catalyzing the phosphorylation of targetproteins. Small molecules may cause cardiotoxicity by: (1) selectivelyinhibiting kinases that also play a role in heart cells (e.g., on-targetside effects); (2) targeting multiple kinases in the same pathway (e.g.,impacting off-target kinases); and (3) inhibiting non-kinase targetsthat play a role in heart function; small molecules may also causecardiotoxicity through a different mechanism. Cardiotoxicity of TK1inhibitors such as imatinib mesylate (Gleevec®), Nilotinib (Tasigna®),sorafenib (Nexavar®), sunitinib (Sutent®) and dasatinib (Sprycel®) hasbeen reported previously (Chu et al., Lancet (2007) 370:2011-2019; Xu etal., Hematol Rev. (2009) Mar. 1; 1(1): e4; Kerketla et al., NatureMedicine (2006) 12:908-916).

Protein kinase activity may also be inhibited by biologic drugs such asmonoclonal antibodies against receptor protein kinases. Thesetherapeutics may exert efficacy by preventing receptor protein kinasesfrom activating and are generally able to bind cell surface antigenswith high specificity. Several monoclonal antibodies target receptorprotein kinases that play an important role in heart function and thusmay cause cardiotoxicity as a result. Trastuzumab and bevacizumab areexamples of monoclonal antibodies that can cause cardiotoxicity (e.g.,heart failure resulting from cardiac tissue damage, electrophysiologicaldysfunction, mitochondrial toxicity, apoptosis, or oxidative stress).Proteasome inhibitor chemotherapy compounds (e.g., bortezomib) are alsoknown to be associated with cardiotoxicity and heart failure.

Currently, the bisdioxopiperazine dexrazoxane (DEX) is the only drugapproved for reducing the incidence of cardiotoxicity and heart failurein cancer patients receiving anticancer agents. Despite its clinicaleffect, DEX is only approved for the treatment of patients withmetastatic breast cancer who have already received accumulated dose of300-500 mg/m² anthracyclines like doxorubicin or epirubicin. DEX is notapproved for use in children and adolescents, it is particularlydisheartening to find reports of high incidences of heart failure inanthracycline-treated young children in their later life of post-cancer.Further, the limited indication approval and use are also testament ofDEX's shortcomings, which include interfering with antitumor efficacy ofanthracyclines, inducing secondary malignancies, and causing blood andbone marrow disorders.

Given the serious impact that many cancer therapies exert on heartfunction, there exists a clear clinical need for developing an effectivedrug that prevents, alleviates, or eliminates cardiotoxicity caused byanthracyclines, protein kinase inhibitors (e.g., tyrosine kinaseinhibitor), proteasome inhibitors and other cancer treatments. Ofparticular importance is the development of drugs that can prevent orreduce cancer drug-induced cardiotoxicity without significantlyinterfering with the anticancer action of the cancer drug. Alsoimportant is the development of cardioprotective drugs that do not causeserious side effects such as neutropenia, worsening of heart problems,or increased risk of secondary malignancies. These potential drugs willsignificantly improve existing cancer therapy, not only by protectingfrom potential heart injuries in cancer patients, but also by enablingchemotherapy doses optimized to achieve maximum-anti-cancer effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 generally depicts a method of reducing cancer treatment-inducedcardiotoxicity in a patient by co-administering a cancer treatment andprotective agent to the patient.

FIG. 2 generally depicts co-administration of a cancer treatment,dexrazoxane (DEX), and a protective agent.

FIG. 3 depicts the effects of mock treatment, doxorubicin (DOX),myricetin, or a co-administration of doxorubicin and myricetin on cellsurvival in human induced pluripotent stem cell-derived cardiomyocytes(iPSC-CM) 3 days following treatment.

FIG. 4A-B depicts the effects of doxorubicin (DOX) (4A), or aco-administration of doxorubicin and myricetin (4B) on mitochondrialhealth in human induced pluripotent stem cell-derived cardiomyocytes(iPSC-CM) 2 days following treatment.

FIG. 5 depicts the effects of mock treatment, doxorubicin, or aco-administration of doxorubicin and myricetin on contractility in humaninduced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) 3 daysfollowing treatment.

FIG. 6A-C depict a chart providing the raw data (6A) or normalized data(6B) for the experiments depicted in FIG. 3 , or the raw data for theexperiments depicted in FIG. 5 (6C).

FIG. 7A-C depict the effects of myricetin (7A), myricitrin/myricetrin(7B), or dihydromyricetin (7C) on doxorubicin-induced apoptosis atincreasing concentrations in human induced pluripotent stem cell-derivedcardiomyocytes (iPSC-CM) at 3 days following treatment.

FIG. 8 is a graph illustrating the protective effect of myricetin (MYR;100 μM) on doxorubicin (DOX)-induced cardiotoxicity at increasingconcentrations of doxorubicin for 72 hours. Y-axis, percentage of cellsurvival; and X-axis, increasing concentrations of DOX.

FIG. 9 is a graph illustrating percentage rescue by increasingconcentrations (X-axis) of myricetin (MYR; circle) and dexrazoxane (DEX;square) of human induced pluripotent stem cell-derived cardiomyocytestreated with 0.5 μM of doxorubicin (DOX).

FIG. 10 depicts the protective effects of myricetin against doxorubicin(DOX)-induced contractility dysfunction in cardiomyocytes, representedin a scale of beat rates (per minute; left panel), duration (in second;center panel) and peak height (in arbituary unit; right panel) for mocktreated, DOX (0.5 μM), DOX plus DEX (100 μM), or DOX plus MYR (100 μM)after 48 hours of treatment.

FIG. 11 depicts the effect of myricetin (MYR) on DOX-induced DNA doublestrand break in human iPSC-derived cardiomyocytes treated with DMSO, DOXalone (0.5 μM), DOX plus DEX (100 μM), or DOX plus MYR (100 μM),measured after 48 hours of the treatment, presented in percentage ofγH2AX-positive cells quantified for each condition (right) andrepresentative images of the cells (left).

FIG. 12 depicts the effect of myricetin (MYR) on doxorubicin(DOX)-induced sarcomere disruption shown in representative images formock treated (DMSO; left), DOX alone (0.5 μM; center), or DOX plus MYR(100 μM; right).

FIG. 13 depicts the effect of myricetin (MYR) on inhibition oftopoisomerases IIα and β (TOPOIIα and TOPOIIβ) compared with that ofdexrazoxane (DEX).

FIG. 14 depicts the effects of myricetin (MYR) and dexrazoxane (DEX) onTOPOIIβ protein degradation illustrated in a graph (top) andrepresentative images (bottom).

FIG. 15 depicts the effect of myricetin (MYR) and dihydromyricetin (DHM)on topoisomerases IIβ (TOPOIIβ) enzymatic inhibition and relativepotency thereof as illustrated in a decatenation gel (top) and a graph(bottom).

FIG. 16 is a graph illustrating relative potency of MYR and DHM inrescuing cardiomyocytes from DOX-induced cell death.

FIG. 17 is a graph illustrating relative potency of MYR and DHM inrescuing cardiomyocytes from DOX-induced double strand break FIG. 18 isa graph illustrating the effect of MYR on RNA expression levels ofTOPOIIα (right) and TOPOIIβ (left) as demonstrated in cardiomyocytestreated with DOX alone or DOX plus MYR.

FIG. 19 depicts two graphs illustrating potency of myricetin (MYR) inprotecting cardiomyocytes from epirubicin (EPI; left) and idarubicin(IDA; right)-induced cytotoxicity.

FIG. 20 is a graph illustrating the effect of myricetin (MYR) onsunitinib (SUN)-induced cell death.

FIG. 21 is a graph illustrating the effect of myricetin (MYR) onsorafenib (SOR)-induced contractile dysfunction.

FIG. 22 is a graph illustrating the effect of myricetin (MYR) onbortezomib (BOR)-induced cell death.

FIG. 23 is a graph illustrating the lack of effect of myricetin (MYR) onDOX's anticancer activity.

FIG. 24 depicts the effect of MYR on DOX-induced contractile dysfunctionin mice measured in percentage of fractional shortening (left) andejection fraction (right).

FIG. 25 depicts the effects of DOX, DEX, and various protective agentson mitochondrial toxicity in cardiomyocytes derived from human inducedpluripotent stem cells.

FIG. 26 depicts the effects of DOX, DEX, and various protective agentson apoptosis in cardiomyocytes derived from human induced pluripotentstem cells.

FIG. 27A-D depict the effects of mock treatment (27A), DEX (27B), aco-administration of doxorubicin and dexrazoxan (27C), or aco-administration of doxorubicin and vitexin (27D), on mitochondrialhealth in human induced pluripotent stem cell-derived cardiomyocytes.

FIG. 28A-B depict the effects of DOX, or co-administration of DOX withvarious concentrations of vitexin (VIT) on the electrophysiologicalactivity in human induced pluripotent stem cell-derived cardiomyocytesover a three-day time period (left), or at a 30-hour time point (right)

FIG. 29A-B depict the effects of co-administration of doxorubicin withkaempferol (KAE; left) and vitexin (VIT; right) on viability inMDA-MB-231 metastatic breast cancer cells.

BRIEF SUMMARY

This disclosure provides composition, kits, and methods for protectingthe heart and for preventing heart failure in patients treated withanthracyclines, protein kinase inhibitors and/or biologic agents. Byminimizing the risk of potentially devastating heart failure in cancerpatient under chemotherapy, conventional cancer treatment can achieveimproved efficacy and safety with the invention described herein.

The compositions include one or more protective agents with or withoutan anticancer agent. The kits often include one or more protectiveagents, and sometimes anticancer agents as well. The methods includemethods of reducing, preventing, or eliminating cardiotoxicity inducedby a drug or other therapy including cancer treatments.

In some aspects, this disclosure provides a pharmaceutical compositioncomprising a protective agent of according to Formula 1,

wherein:

X¹ is CR⁵R⁶, NR⁵, O, S, C═O, or C═S;

each of R¹, R², R³, R⁵, R⁶, R⁹, and R¹⁰ is independently alkyl, alkenyl,alkynyl, alkoxy, acyl, acyloxy, carboxylic acid, ester, amine, amide,carbonate, carbamate, nitro, thioether, thioester, cycloalkyl,heteroalkyl, heterocyclyl, monosaccharide, aryl, or heteroaryl, any ofwhich is substituted or unsubstituted, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, or H;

R⁴, R⁷ and R⁸ are alkoxy, hydroxyl or H;

W¹ is O or S, or

a salt thereof.

In some aspects, X¹ can be O or S; each of R¹, R², R³, R⁹, and R¹⁰ canbe independently alkoxy, cycloalkyl, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, or H; and each of R⁴, R⁷ and R⁸ can beindependently alkoxy, hydroxyl or H.

In some aspects, X¹ is O; each of R¹, R², R³, R⁹, and R¹⁰ can beindependently alkoxy, cycloalkyl, halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, or H; and each of R⁴, R⁷ and R⁸ can beindependently alkoxy, hydroxyl or H.

In yet other aspects, X¹ is O; each of R¹ and R² can be independentlyhydroxyl or H; each of R³, R⁹ and R¹⁰ can be independently cycloalkyl,heterocyclyl, hydroxyl, or H; R⁴ is hydroxyl; and each of R⁷ and R⁸ canbe independently hydroxyl or H.

In yet other aspects, X¹ is O; R¹ is hydroxyl; each of R² and R³ can beindependently hydroxyl or H; R⁹ and R¹⁰ are H; R⁴ is hydroxyl; and eachof R⁷ and R⁸ can be independently hydroxyl or H.

In yet other aspects, X¹ is O; R¹ is hydroxyl; each of R² and R³ can beindependently hydroxyl or H; R⁹ can be heterocyclyl or H; of R¹⁰ is H;R⁴ can be independently hydroxyl or H; and each of R⁷ and R⁸ can beindependently hydroxyl or H.

In yet other aspects, X¹ is O; R¹ is hydroxyl; each of R² and R⁹ can beindependently hydroxyl or H; R³ can be cycloalkyl, hydroxyl or H; R¹⁰ isH; R4 is hydroxyl; and each of R⁷ and R⁸ can be independently hydroxylor H. In one embodiment, cycloalkyl of R³ can be a monosaccharide.

In some embodiments, the pharmaceutical composition may comprisemyricetin and is a compound according to the following formula.

In some embodiments, the pharmaceutical composition may comprisemyricetrin/myricitrin and is a compound according to the followingformula.

In some embodiments, the pharmaceutical composition may compriserobinetin and is a compound according to the following formula.

In some embodiments, the pharmaceutical composition may comprisetricetin and is a compound according to the following formula.

In some embodiments, the pharmaceutical composition may comprise7,3′,4′,5′-tetrahydroxyflavone and is a compound according to thefollowing formula.

In some embodiments, the pharmaceutical composition comprises ficetin.In some embodiments, the pharmaceutical composition comprises quercetin.In some embodiments, the pharmaceutical composition compriseskaempferol. In some embodiments, the protective agent within thepharmaceutical composition can be a compound with the followingstructure:

In a particular example, the protective agent within the pharmaceuticalcomposition can be vitexin.

In some embodiments, the pharmaceutical composition may include one ormore chemotherapy drug(s) (anticancer agent) or biologic agent(s). Insome embodiments, the pharmaceutical composition can include achemotherapy drug. In some embodiments, the pharmaceutical compositionmay include one or more chemotherapy drug(s) (anticancer agent) and oneor more of the protective agent(s) selected from the group consisting ofmyricetin, tricetin (5,7,3′,4′,5′-pentahydroxyflavone), robinetin,ficetin, vitexin, 7,3′,4′,5′-tetrahydroxyflavone, and myricetrin.

In some embodiments, the pharmaceutical composition may comprise ananthracycline or salt thereof. In some embodiments, the anthracyclinecan be daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone,or valrubicin. In some embodiments, the anthracycline is doxorubicin. Insome embodiments, the anthracycline is epirubicin. In some embodiments,the anthracycline is idarubicin.

In some embodiments, the chemotherapy drug can be a protein kinaseinhibitor. In some embodiments, the protein kinase inhibitor isafatinib, axitinib, bosutinib, cabozantinib, carfilzomib, ceritinib,cobimetanib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus,gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,nintedanib, osimertinib, palbociclib, pazopanib, pegaptanib, ponatinib,regorafenib, ruxolitinib, sirolimus, sorafenib, sunitinib, tofacitinib,tofacitinib, temsirolimus, trametinib, vandetanib, vemurafenib, orvismodegib.

In some embodiments, the chemotherapy drug can be a proteasomeinhibitor. In a particularly example, the proteasome inhibitor can bebortezomib.

In some embodiments, the protein kinase inhibitor can be a tyrosinekinase inhibitor. In some embodiments, for example, the tyrosine kinaseinhibitor is selected from the group consisting of sorafenib, sunitinib,bosutinib, gefitinib, dasatinib, dabrafenib, vemurafenib, imatinib,lapatinib, mesylate, and nilotinib. In a particular example, thetyrosine kinase inhibitor is sorafenib. In another particular example,the tyrosine kinase inhibitor is sunitinib.

In some embodiments, the chemotherapy drug can be a biologic agent. Insome embodiments, the biologic agent is an antibody. In someembodiments, the antibody can be adotrastuzumabemtansine, alemtuzumab,bevacizumab, blinatumomab, brentuximab vedotin, catumaxomab, cetuximab,gemtuzumab ozogamicin, ibritumomab tiuxetan, ipilimumab, necitumumab,nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab,pertuzumab, ramucirumab, rituximab, tositumomab-I131, or trastuzumab. Inone particular example, the antibody is trastuzumab.

In some embodiments, the pharmaceutical composition can be a liquidcomposition. In some embodiments, the pharmaceutical composition can bea capsule, a gel capsule, or a liposome. In some embodiments, thepharmaceutical composition can be a tablet.

In some embodiments, the pharmaceutical composition may also includedexrazoxane as an additional protective agent.

In some embodiments, the pharmaceutical composition can comprise atleast 1 mg of one or more protective agents. In some embodiments, thepharmaceutical composition can comprise between 0.1 mg and 200 mg of oneor more protective agents. In some embodiments, the pharmaceuticalcomposition can comprise between 0.1 mg and 300 mg of one or moreprotective agents.

In some embodiments, two protective agents are present and co-formulatedtogether. In some embodiments, the two protective agents can be presentas distinct entities within the pharmaceutical composition. In someembodiments, the pharmaceutical composition can comprise thechemotherapy drug and the chemotherapy drug is co-formulated with one ofthe two protective agents.

In some aspects, this disclosure provides a pharmaceutical compositioncomprising (a) a protective agent selected from the group consisting of:

a compound according to Formula 2,

wherein:

represents a single or double bond;X¹ is CR⁵R⁶, NR⁵, O, S, C═O, or C═S;each of R¹, R², R³, R⁵, R⁶, R⁹, and R¹⁰ is independently alkyl, alkenyl,alkynyl, alkoxy, acyl, acyloxy, carboxylic acid, ester, amine, amide,carbonate, carbamate, nitro, thioether, thioester, cycloalkyl,heteroalkyl, heterocyclyl, aryl, or heteroaryl, any of which issubstituted or unsubstituted, halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, or H;R⁴, R⁷ and R⁸ are hydroxyl;W¹ is O or S;or a salt thereof, and(b) a chemotherapy drug or a biologic agent.

In some embodiments, the pharmaceutical composition may comprise ananticancer agent or a chemotherapy drug. In some embodiments, theprotective agent is selected from the group consisting of myricetin,tricetin, robinetin, ficetin, vitexin, dihydrorobinetin,7,3′,4′,5′-tetrahydroxyflavone, and myricetrin.

In some embodiments, the pharmaceutical composition may comprise one ormore protective agents. In some embodiments, the pharmaceuticalcomposition may comprise myricetin. In some embodiments, thepharmaceutical composition may comprise myricetrin. In some embodiments,the pharmaceutical composition may comprise robinetin. In someembodiments, the pharmaceutical composition may comprisedihydrorobinetin. In some embodiments, the pharmaceutical compositionmay comprise vitexin. In some embodiments, the pharmaceuticalcomposition may comprise tricetin. In some embodiments, thepharmaceutical composition comprises quercetin. In some embodiments, thepharmaceutical composition comprises kaempferol.

In some embodiments, the pharmaceutical composition comprises ananthracycline or salt thereof. In some embodiments, the anthracycline isdaunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, orvalrubicin. In some embodiments, the anthracycline is doxorubicin. Insome embodiments, the anthracycline is epirubicin. In some embodiments,the anthracycline is idarubicin.

In some embodiments, the chemotherapy drug can be a protein kinaseinhibitor. In some embodiments, the protein kinase inhibitor isafatinib, axitinib, bosutinib, cabozantinib, carfilzomib, ceritinib,cobimetanib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus,gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,nintedanib, osimertinib, palbociclib, pazopanib, pegaptanib, ponatinib,regorafenib, ruxolitinib, sirolimus, sorafenib, sunitinib, tofacitinib,tofacitinib, temsirolimus, trametinib, vandetanib, vemurafenib, orvismodegib.

In some embodiments, the chemotherapy drug is a proteasome inhibitor. Ina particular example, the proteasome inhibitor is bortezomib.

In some embodiments, the protein kinase inhibitor is a tyrosine kinaseinhibitor. In some embodiments, the tyrosine kinase inhibitor isselected from the group consisting of sorafenib, sunitinib, bosutinib,gefitinib, dasatinib, dabrafenib, vemurafenib, imatinib, lapatinib,mesylate, and nilotinib. In a particular example, the tyrosine kinaseinhibitor is sorafenib. In another particular example, the tyrosinekinase inhibitor is sunitinib.

In some embodiments, the chemotherapy drug is a biologic agent. In someembodiments, the biologic agent is an antibody. In some embodiments, theantibody is adotrastuzumabemtansine, alemtuzumab, bevacizumab,blinatumomab, brentuximab vedotin, catumaxomab, cetuximab, gemtuzumabozogamicin, ibritumomab tiuxetan, ipilimumab, necitumumab, nivolumab,obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab,ramucirumab, rituximab, tositumomab-I131, or trastuzumab. In aparticular example, the antibody is trastuzumab. In a particularexample, the antibody is bevacizumab.

In some embodiments, the pharmaceutical composition may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, or 300 mg of one or more protectiveagents.

In some embodiments, the pharmaceutical composition may comprise between0.1 mg and 50 mg of the protective agent. In some embodiments, thepharmaceutical composition may comprise between 1 mg and 10 mg of theprotective agent. In some embodiments, the pharmaceutical compositionmay comprise between 1 mg and 20 mg of the protective agent. In someembodiments, the pharmaceutical composition may comprise between 1 mgand 30 mg of the protective agent. In some embodiments, thepharmaceutical composition may comprise between 1 mg and 40 mg of theprotective agent. In some embodiments, the pharmaceutical compositionmay comprise between 1 mg and 50 mg of the protective agent. In someembodiments, the pharmaceutical composition may comprise between 1 mgand 100 mg of the protective agent. In some embodiments, thepharmaceutical composition may comprise between 1 mg and 200 mg of theprotective agent. In some embodiments, the pharmaceutical compositionmay comprise between 40 mg and 300 mg of the protective agent. In someembodiments, the pharmaceutical composition may comprise between 50 mgand 400 mg of the protective agent.

In some embodiments, the pharmaceutical composition may comprise thechemotherapy drug, and the chemotherapy drug and the protective agentare mixed within the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises thechemotherapy drug wherein the dose of the chemotherapy drug is at least0.1 mg. In some embodiments, the pharmaceutical composition comprisesthe chemotherapy drug wherein the dose of the chemotherapy drug isbetween 0.01 mg and 50 mg. In some embodiments, the pharmaceuticalcomposition comprises the chemotherapy drug wherein the dose of thechemotherapy drug is between 0.01 mg and 100 mg. In some embodiments,the pharmaceutical composition comprises the chemotherapy drug whereinthe dose of the chemotherapy drug is between 0.01 mg and 200 mg.

In some embodiments, the pharmaceutical composition comprises thebiologic agent at a dose of at least 50 mg. In some embodiments, thepharmaceutical composition comprises a biologic agent at a dose ofbetween 0.1 mg and 100 mg. In some embodiments, the pharmaceuticalcomposition comprises a biologic agent at a dose of between 0.1 mg and200 mg.

In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 1:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and a molarratio of the protective agent to the chemotherapy drug is at least 2:1.In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 3:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and a molarratio of the protective agent to the chemotherapy drug is at least 4:1.In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 5:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and a molarratio of the protective agent to the chemotherapy drug is at least 6:1.In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 7:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and a molarratio of the protective agent to the chemotherapy drug is at least 8:1.In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 9:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and a molarratio of the protective agent to the chemotherapy drug is at least 10:1.In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and a molar ratio of the protective agent to thechemotherapy drug is at least 20:1. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and wherein amolar ratio of the protective agent to the chemotherapy drug is at least100:1. In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and wherein a molar ratio of the protective agent tothe chemotherapy drug is at least 1:2. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and wherein amolar ratio of the protective agent to the chemotherapy drug is at least1:3. In some embodiments, the pharmaceutical composition comprises thechemotherapy drug and wherein a molar ratio of the protective agent tothe chemotherapy drug is at least 1.4. In some embodiments, thepharmaceutical composition comprises the chemotherapy drug and wherein amolar ratio of the protective agent to the chemotherapy drug is at least1:5.

This disclosure provides methods for administering to a subject any ofthe pharmaceutical compositions disclosed herein. In some aspects, thisdisclosure provides a method for preventing, reducing, or eliminatingcardiotoxicity or heart failure in general. In some aspects, thisdisclosure provides a method for preventing, reducing, or eliminatingcardiotoxicity induced by a chemotherapy drug or biologic agent in asubject, the method comprising: administering one or more protectiveagent according to Formula 1, to the subject, thereby preventing,reducing, or eliminating the cardiotoxicity induced by the chemotherapydrug or biologic agent in the subject. In some cases, the pharmaceuticalcomposition comprises a compound selected from the group consisting ofsuch as myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, and myricitrin.

In some aspects, this disclosure provides a method for preventing,reducing, or eliminating cardiotoxicity induced by a chemotherapy drugor biologic agent in a subject, the method comprising: administering atleast one protective agent according to Formula 1 or Formula 2, to thesubject, thereby preventing, reducing, or eliminating the cardiotoxicityinduced by the chemotherapy drug or biologic agent in the subject.

In some embodiments, the subject is administered a chemotherapy drug orbiologic agent prior to the administering of one or more protectiveagent(s) according Formula 1 or 2, to the subject.

In some embodiments, the subject is administered a chemotherapy drug orbiologic agent following the administering of at least two protectiveagents of Formula 1 or 2 to the subject.

In some aspects, this disclosure provides a method for treating cancer,the method comprising: (a) administering a chemotherapy drug or biologicagent to a subject, wherein the subject has cancer and the chemotherapydrug or biologic agent is capable of causing cardiotoxicity in thesubject; and (b) administering at least one protective agent accordingto Formula 1 or Formula 2 to the subject, wherein the protective agentprevents, reduces, or eliminates the cardiotoxicity in the subject.

In some embodiments, the subject has a human suffering from cancer. Insome embodiments, the cancer is bladder cancer, bone cancer, a braintumor, breast cancer, esophageal cancer, gastrointestinal cancer,leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer,prostate cancer, a sarcoma, stomach cancer, or thyroid cancer.

In some embodiments, prior to the administration of the protectiveagent, the subject has a cardiac condition or has a history of having acardiac condition. In some embodiments, the administration of theprotective agent reduces the risk of the subject experiencingcardiotoxicity induced by the chemotherapy drug or biologic agent. Insome embodiments, the administration of the protective agent reduces therisk of the subject experiencing cardiotoxicity induced by thechemotherapy drug or biologic agent by at least 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95%, In some embodiments, the cardiotoxicity may comprisecardiac tissue damage, electrophysiological dysfunction, mitochondrialtoxicity, apoptosis, or oxidative stress. In some embodiments, thecardiotoxicity is cardiac tissue damage. In some embodiments, thecardiotoxicity is electrophysiological dysfunction.

In some embodiments, the chemotherapy drug used in the methods describedherein may comprise an anthracycline or a salt thereof. In someembodiments, the anthracycline is daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, or valrubicin. In some embodiments, theanthracycline is doxorubicin. In some embodiments, the anthracycline isepirubicin. In some embodiments, the anthracycline is idarubicin.

In some embodiments, the chemotherapy drug used in the methods describedherein is a protein kinase inhibitor. In some embodiments, the proteinkinase inhibitor is afatinib, axitinib, bosutinib, cabozantinib,carfilzomib, ceritinib, cobimetanib, crizotinib, dabrafenib, dasatinib,erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib,lenvatinib, nilotinib, nintedanib, osimertinib, palbociclib, pazopanib,pegaptanib, ponatinib, regorafenib, ruxolitinib, sirolimus, sorafenib,sunitinib, tofacitinib, tofacitinib, temsirolimus, trametinib,vandetanib, vemurafenib, or vismodegib.

In some embodiments, the protein kinase inhibitor is a tyrosine kinaseinhibitor. In some embodiments, the protein kinase inhibitor is atyrosine kinase inhibitor. In some embodiments, the tyrosine kinaseinhibitor is selected from the group consisting of sorafenib, sunitinib,bosutinib, gefitinib, dasatinib, dabrafenib, vemurafenib, imatinib,lapatinib, mesylate, and nilotinib. In a particular example, thetyrosine kinase inhibitor is sorafenib. In another particular example,the tyrosine kinase inhibitor is sunitinib.

In some aspect, the chemotherapy drug is a proteasome inhibitor. In oneparticular example, the proteasome inhibitor is bortezomib.

In some embodiments, the biologic agent used in the methods describedherein can be an antibody. In some embodiments, the antibody isadotrastuzumabemtansine, alemtuzumab, bevacizumab, blinatumomab,brentuximab vedotin, catumaxomab, cetuximab, gemtuzumab ozogamicin,ibritumomab tiuxetan, ipilimumab, necitumumab, nivolumab, obinutuzumab,ofatumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab,rituximab, tositumomab-I131, or trastuzumab. In one particular example,the antibody is trastuzumab.

In some embodiments, the subject according to the methods describedherein has a decreased QTc interval after administering the protectiveagent. In some cases, the protective agent is selected from the groupconsisting of such as myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, and myricitrin. In one particularexample, the protective agent is myricetin.

In some embodiments, the chemotherapy drug and protective agent ofFormula 1 or Formula 2 are administered concurrently to the subject. Insome embodiments, the chemotherapy drug and protective agent areadministered sequentially to the subject. In some embodiments, theprotective agent is administered to the subject prior to theadministration of the chemotherapy drug. In some embodiments, theprotective agent is administered to the subject after the administrationof the chemotherapy drug.

In some embodiments, at least two protective agents of Formula 1 orFormula 2 can be administered. For example, the at least two protectiveagents can be selected from the group consisting of such as myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,dihydrorobinetin, and myricitrin. In some embodiments, one or moreprotective agent(s) can further comprise dexrazoxane.

This disclosure provides a method for treating or preventing organdamage in a subject comprising: administering one or more protectiveagents selected from the group consisting of such as myricetin, vitexin,robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone, andmyricitrin to a subject with organ damage, thereby treating orpreventing organ damage in the subject.

This disclosure also provides kits. In some aspects, this disclosureprovides a kit comprising: (a) a protective agent selected from thegroup consisting of myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, and myricitrin; and (b) a chemotherapydrug or a biologic agent.

In some aspects, this disclosure provides a kit comprising: (a) aprotective agent selected from the group consisting of myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,and myricitrin; (b) a chemotherapy drug or a biologic agent; and (c)dexrazoxane. In some embodiment, the protective agent is myricetin.

DETAILED DESCRIPTION

Certain cancer drugs (e.g., anthracycline drugs, protein kinaseinhibitors) and other therapies can cause cardiotoxicity in patients.For example, anthracycline-induced cardiotoxicity occurs when the drugsuch as doxorubicin intercalates the DNA upon a cleavage of DNA bytopoisomerase II enzymes thereby effectively preventing TOPOIIα or βfrom ligating the cleaved strands back together.

This disclosure provides pharmaceutical compositions and methods thatmay prevent, reduce or eliminate such cardiotoxicity and that may alsoprevent, reduce or eliminate organ damage caused by cardiac tissuedamage, electrophysiological dysfunction, mitochondrial toxicity,apoptosis, or oxidative stress. Many of the compositions and methodsprovided herein relate to the administration of a specific protectiveagent in conjunction with one or more cancer treatments, therebyreducing the risk that the cancer treatment will cause or aggravatecardiotoxic events in a patient. The protective agents described hereininclude such as myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrin and/orderivatives or salts thereof. In some cases, the protective agents maybe flavonoids. In some cases, the protective agent may be administeredin combination with a different protective agent. In some cases, theprotective agent may be administered in combinations such ascombinations including dexrazoxane and another protective agent.

The present disclosure may enable cancer patients—including hearthealthy patients and patients with pre-existing cardiac conditions—toreceive a desired dosage of a therapy (e.g., an anthracycline or saltthereof) without having the dosage regimen significantly altered by therisk of cardiotoxicity. Another advantage of the present disclosure isthat it may enable a larger patient population to receive a giventherapy, such as certain patients with pre-existing cardiac conditionsor with age limits. In addition, the reduction or prevention ofcardiotoxicity may enable a cancer patient to avoid having to take amedication to treat a heart condition. Overall, the advantages presentedherein may help to facilitate a better therapeutic outcome for patients.

The pharmaceutical compositions and methods (including methods of use)provided herein generally relate to reducing, eliminating or preventingcardiotoxicity caused by chemotherapeutic drugs, biologic agents, orradiation therapy; they can also be used to reduce or eliminate organdamage caused by electrophysiological dysfunction, mitochondrialtoxicity, apoptosis, or oxidative stress. FIG. 1 depicts a generalschematic of some embodiments of the methods provided herein. The toppanel shows a cancer treatment [110], such as a chemotherapeutic drug,biologic agent, or radiation therapy, being administered to a patient[120], who develops cardiotoxicity and is then gradually given reduceddoses of the cancer treatment over time [130]. Therefore, thecardiotoxicity associated with administration of the cancer treatment[110] in the absence of a protective agent [140] may limit the patientpopulation that is eligible to receive treatment. In the bottom panel,the cancer treatment [110] is co-administered with a protective agent[140], such as myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin and myricitrin to apatient, e.g., [151] who experiences reduced cardiotoxicity, or nocardiotoxicity at all [160], thereby enabling the patient to toleratethe dosage regimen. Although separate vehicles for the cancer treatmentand protective agent are depicted, in some cases the cancer treatmentand protective agent are co-formulated together. The co-administrationof the cancer treatment [110] with the protective agent [140] may enablea larger patient population [150] to receive the cancer treatment,including healthy patients and patients with pre-existing cardiacconditions [152, 153].

FIG. 2 also depicts a general schematic of embodiments provided herein.The top panel shows a cancer treatment [210] (e.g., a chemotherapeuticdrug, a biologic agent, or radiation therapy), and dexrazoxane [220]being co-administered to a patient [230] who then experiences somecardiotoxicity over time [240]. The co-administration of the cancertreatment [210] and dexrazoxane [220] in the absence of the protectiveagent [250] may limit the patient population that is eligible to receivetreatment. In the bottom panel, the cancer treatment [210], thedexrazoxane [220], and a protective agent [250] (such as myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,dihydrorobinetin, and myricitrin) are administered to a patient [261]who experiences reduced cardiotoxicity, or no cardiotoxicity at all[270]. In this embodiment, the co-administration of the protective agent[250] with the cancer treatment [210] and dexrazoxane [220] may enhancethe activity of dexrazoxane to prevent, alleviate, or eliminatecardiotoxicity in a patient [261], thereby enabling a larger patientpopulation [260] to receive treatment, including patients without andthose with pre-existing cardiac conditions [262, 263]. In some cases,the protective agent, the dexrazoxane and/or the cancer treatment areadministered separately; in some cases, they are administeredconcurrently or as co-formulations. Generally, the co-formulations andmethods provided herein may reduce the cardiotoxicity induced inpatients by chemotherapeutic drugs, biologic agents, or radiationtherapy.

The compositions provided herein may include a co-formulation of two ormore protective agents. For example, the co-formulation may comprisesuch as myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrin, anddexrazoxane. In some cases, the compositions may include aco-formulation of a protective agent (e.g., such as myricetin, vitexin,robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,dihydrorobinetin, and myricitrin) with a certain cancer treatment (e.g.,chemotherapeutic drug or biologic agent). In some cases, provided hereinare kits that contain at least two protective agents (or a protectiveagent and a cancer treatment) as separate components, often along withinstructions for use.

Methods

Provided herein are methods for administering to a patient, particularlya cancer patient, a pharmaceutical composition that can reduce,eliminate or prevent cardiotoxicity caused by a cancer treatment (e.g.,chemotherapeutic drugs, biologic agents or radiation therapy). Themethods provided herein also comprise treating cancer in a patient usingat least one of the compositions provided herein. In some cases, thepatient may be heart-healthy; in some cases, the patient is at-risk fora cardiac condition.

The methods provided herein generally comprise administering to apatient a pharmaceutical composition comprising at least one protectiveagent described herein, or at least one protective agent and a cancertreatment (e.g., anthracycline drug, protein kinase inhibitor, biologicagent, or radiation therapy). The protective agent and cancer treatmentmay also be combined with a different cardioprotective agent (e.g.,dexrazoxane). In some cases, the protective agent and cancer treatmentmay be co-formulated, in that they are mixed within the samepharmaceutical composition (e.g., tablet, capsule, liposome, liquid, orvapor); in some cases, they exist as distinct entities.

Subjects

The methods and compositions disclosed herein are generally used toprevent, reduce, treat, or eliminate cancer treatment-inducedcardiotoxicity in a subject. The subject may be any human patient,particularly a cancer patient, a patient at risk for cancer, or apatient with a family or personal history of cancer. In some cases, thepatient is in a particular stage of cancer treatment. For example, apharmaceutical composition described herein can be administered to ahuman patient with early or late stage cancer in order to reducecardiotoxicity caused by a cancer treatment.

The cancer patients may have any type of cancer. Examples of cancer caninclude, but are not limited to, adrenal cancer, anal cancer, basal cellcarcinoma, bile duct cancer, bladder cancer, cancer of the blood, bonecancer, a brain tumor, breast cancer, bronchus cancer, cancer of thecardiovascular system, cervical cancer, colon cancer, colorectal cancer,cancer of the digestive system, cancer of the endocrine system,endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, agastrointestinal tumor, kidney cancer, hematopoietic malignancy,laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma,melanoma, mesothelioma, cancer of the muscular system, MyelodysplasticSyndrome (MDS), myeloma, nasal cavity cancer, nasopharyngeal cancer,cancer of the nervous system, cancer of the lymphatic system, oralcancer, oropharyngeal cancer, osteosarcoma, Kaposi sarcoma, ovariancancer, pancreatic cancer, penile cancer, pituitary tumors, prostatecancer, rectal cancer, renal pelvis cancer, cancer of the reproductivesystem, cancer of the respiratory system, sarcoma, salivary glandcancer, skeletal system cancer, skin cancer, small intestine cancer,stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroidcancer, a tumor, cancer of the urinary system, uterine cancer, vaginalcancer, or vulvar cancer. The term ‘lymphoma’ may refer to any type oflymphoma including B-cell lymphoma (e.g., diffuse large B-cell lymphoma,follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma,marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma, hairy cell leukemia, or primary central nervous systemlymphoma) or a T-cell lymphoma (e.g., precursor T-lymphoblasticlymphoma, or peripheral T-cell lymphoma). The term ‘leukemia’ may referto any type of leukemia including acute leukemia or chronic leukemia.Types of leukemia include acute myeloid leukemia, chronic myeloidleukemia, acute lymphocytic leukemia, acute undifferentiated leukemia,or chronic lymphocytic leukemia. In some cases, the cancer patient doesnot have a particular type of cancer. For example, in some instances,the patient may have a cancer that is not breast cancer.

Examples of cancer include cancers that cause solid tumors as well ascancers that do not cause solid tumors. Furthermore, any of the cancersmentioned herein may be a primary cancer (e.g., a cancer that is namedafter the part of the body where it first started to grow) or asecondary or metastatic cancer (e.g., a cancer that has originated fromanother part of the body).

A patient at risk of cancer may be at risk because of a particularcondition such as a pre-cancerous condition. Pre-cancerous conditionsinclude but are not limited to actinic keratosis, Barrett's esophagus,atrophic gastritis, ductal carcinoma in situ, dyskeratosis congenita,sideropenic dysphagia, lichen planus, oral submucous fibrosis, solarelastosis, cervical dysplasia, leukoplakia, and erythroplakia). In somecases, a patient may be at risk of cancer because of cell or tissuedysplasia (e.g., an abnormal change in cell number, abnormal change incell shape, abnormal change in cell size, or abnormal change in cellpigmentation).

A patient at risk of cancer may be a patient that was exposed to acarcinogenic agent. Such patients may include patients with exposure toknown or probable carcinogens (e.g., acetyl aldehyde, asbestos, ortobacco products), or patients exposed to ionizing radiation (e.g.,gamma radiation, beta-radiation, X-radiation, or ultraviolet radiation).In some cases, a patient at risk of cancer is at risk because of afamily history of cancer.

The methods and compositions disclosed herein may also be used toprevent, reduce, or eliminate cardiotoxicity in patients with a historyof cancer, particularly patients who have been administered cancertreatments (e.g., anthracycline drugs, protein kinase inhibitors,proteasome inhibitors, or biological agents) with cardiotoxic effectsExamples of a patient with a history of cancer include, but are notlimited to, a patient in remission, a patient in complete remission, apatient with relapsed cancer or a patient with recurring cancer.

The methods and compositions disclosed herein are generally used in apatient that has been administered, or is currently being administered,a cardiotoxicity-inducing agent (e.g., a cancer treatment). Non-limitingexamples of cardiotoxicity-inducing agents are described elsewhereherein and may include cancer treatments, chemotherapeutic drugs,anthracyclines (e.g., doxorubicin, epirubicin, and idarubicin), proteinkinase inhibitors (e.g., tyrosine kinase inhibitor), biologic agents(e.g., trastuzumab), or radiation therapy, as well as any cancertreatment otherwise known to cause cardiotoxicity. In some examples, apharmaceutical composition disclosed herein is administered to a cancerpatient with previous exposure to a cancer treatment known to havecardiotoxic effects, in order to reduce the risk of cardiotoxicityassociated with the patient's current cancer treatment regimen. In somecases, the pharmaceutical composition is administered to a cancerpatient in order to reduce or off-set cumulative effects of priorexposures to cancer treatment or drugs, or to other agents that causecardiotoxicity. In some examples, a pharmaceutical co-formulationcomprising myricetin and anthracycline may be administered to a prostatecancer patient who also has dilated cardiomyopathy caused by a previouscancer treatment. In another example, a pharmaceutical co-formulationcomprising vitexin may be administered to a lung cancer patient who isbeing concurrently treated with an anthracycline. In yet anotherexample, a pharmaceutical co-formulation comprising robinetin may beadministered to a breast cancer patient. In yet another example, apharmaceutical co-formulation comprising tricetin may be administered toa Kaposi sarcoma cancer patient. In yet another example, apharmaceutical co-formulation comprising ficetin may be administered toa breast cancer patient. In yet another example, a pharmaceuticalco-formulation comprising 7,3′,4′,5′-tetrahydroxyflavone may beadministered to a breast cancer patient. In yet another example, apharmaceutical co-formulation comprising myricitrin may be administeredto a breast cancer patient. In yet another example, a pharmaceuticalco-formulation comprising myricetin and anthracycline may beadministered to a liver cancer patient who also has dilatedcardiomyopathy caused by a previous cancer treatment. In yet anotherexample, a pharmaceutical co-formulation comprising myricetin anddoxorubicin may be administered to a sarcoma cancer patient.

In some cases, the methods and compositions herein may be used toalleviate cardiotoxicity that is not caused by a cancer treatment. Assuch, the patient may have or be at risk of having, cardiotoxicityinduced by a drug that is not specifically for cancer, such as a proteinkinase inhibitor. Such patients may have a condition such as aneurological or cardiac disorder. In some cases, the condition may be acondition treatable by a protein kinase inhibitor.

In some cases, the patient may have organ damage or be at risk of havingorgan damage. For example, the patient may have organ damage (or be atrisk of organ damage) as a result of cardiac tissue damage,electrophysiological dysfunction, mitochondrial toxicity, apoptosis, oroxidative stress. For such patients, the methods and compositionsprovided herein may reduce or eliminate the organ damage caused bycardiac tissue damage, electrophysiological dysfunction, mitochondrialtoxicity, apoptosis, or oxidative stress.

In some cases, patients treated by any of the methods or compositionsdescribed herein may have heart disease, or have a family history ofheart disease. Examples of heart disease include, but are not limitedto, arrhythmogenic cardiomyopathy, arterial disease, Brugada Syndrome,congenital heart disease, dilated cardiomyopathy, heart palpitations,heart valve disease, hypertensive heart disease, hypertrophiccardiomyopathy, long QT syndrome, rheumatic heart disease, or vasculardisease. In some cases, the heart disease is caused by a cardiotoxicagent (e.g., anthracycline). For example, the heart disease may becaused by any of the cardiotoxic agents mentioned herein. In oneparticular example, a pharmaceutical co-formulation comprising myricetinand doxorubicin may be administered to a breast cancer patient who alsohas hypertrophic cardiomyopathy. In another example, a co-formulation ofone or more of compound selected from the group consisting of myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,dihydrorobinetin, and myricitrin may be administered to a cancer patientexperiencing cardiotoxicity from a previously administered chemotherapydrug.

A patient treated by any of the methods or compositions described hereinmay be of any age and may be an adult, infant or child. In some cases,the patient is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 years old, or within arange therein (e.g., between 2 and 20 years old, between 20 and 40 yearsold, or between 40 and 90 years old). A particular class of patientsthat may benefit is patients over the age of 40. Another particularclass of patients that may benefit is pediatric patients, who may be atlife risk of chronic heart symptoms. Furthermore, a patient treated byany of the methods or compositions described herein may be male orfemale.

Any of the compositions disclosed herein may also be administered to anon-human subject, such as a laboratory or farm animal. Non-limitingexamples of a non-human subject include a dog, a goat, a guinea pig, ahamster, a mouse, a pig, a non-human primate (e.g., a gorilla, an ape,an orangutan, a lemur, or a baboon), a rat, a sheep, a cow, or azebrafish.

Drug Administration

The disclosure provided herein describes methods to prevent, reduce, oreliminate cancer treatment-induced cardiotoxicity in patients byadministering to a patient one or more protective agents of Formula 1,Formula 2 or derivative or salt thereof. The disclosure herein alsodescribes methods to prevent, reduce, or eliminate cancertreatment-induced cardiotoxicity in patients by administering to apatient one or more protective agent selected from the group consistingof myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, and myricitrin (orderivative or salt thereof). The disclosure provided herein alsodescribes methods of administering to a subject, wherein the subject hascancer and the cancer treatment is capable of causing cardiotoxicity andorgan damage in the subject, and administering one or more protectiveagents (or derivative or salt thereof) selected from the groupconsisting of myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, and myricitrin,wherein the protective agent prevents, reduces, or eliminates thecardiotoxicity in the subject.

Methods disclosed herein can further comprise administering to thepatient a combination of dexrazoxane (or derivative or salt thereof) anda protective agent according to Formula 1, Formula 2, or derivative orsalt thereof; the combined agents may be administered as aco-formulation or separately. In some aspects, the methods compriseadministering to the patient a combination of dexrazoxane (or derivativeor salt thereof) and myricetrin (or derivative or salt thereof); thecombined agents may be administered as a co-formulation or separately.

Methods disclosed herein can further comprise administering to thepatient combined agents comprising a combination of dexrazoxane (orderivative or salt thereof) and a protective agent selected from thegroup consisting of myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, and myricitrin. (orderivative or salt thereof); the combined agents may be administered asa co-formulation or separately.

The protective agents may be administered to the subject or patient inany combination of a compound of Formula 1 or Formula 2. In some cases,only one protective agent (e.g., myricetin or a derivative or saltthereof) is administered to a subject or patient. In some cases, onlyone protective agent (e.g., myricitrin or a derivative or salt thereof)is administered to a subject or patient. In some cases, only oneprotective agent (e.g., vitexin or a derivative or salt thereof) isadministered to a subject or patient. In some cases, only one protectiveagent (e.g., robinetin or a derivative or salt thereof) is administeredto a subject or patient. In some cases, only one protective agent (e.g.,tricetin or a derivative or salt thereof) is administered to a subjector patient. In some cases, only one protective agent (e.g.,7,3′,4′,5′-tetrahydroxyflavone or a derivative or salt thereof) isadministered to a subject or patient. In a particular example, a subjector patient described herein may be administered a therapeuticallyeffective dose of myricetin (or derivative or salt thereof). In anotherexample, a subject or patient described herein may be administered atherapeutically effective dose of robinetin (or derivative or saltthereof). In yet another example, a subject or patient described hereinmay be administered a therapeutically effective dose of vitexin (orderivative or salt thereof).

In some cases, two protective agents (or derivative or salt thereof)selected from the group consisting of myricetin, vitexin, robinetin,tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin,myricitrin, and dexrazoxane are administered to a subject. In caseswhere two or more protective agents are administered to a patient, theprotective agents may be administered as distinct entities or in aco-formulation. For example, a patient experiencing cardiotoxicity maybe administered a therapeutically effective co-formulation of myricetinand robinetin; myricetin and dexrazoxan; or other co-formulationdescribed herein. The two or more protective agents may be administeredsimultaneously or sequentially. In some cases, the two or moreprotective agents may be administered sequentially in a particularorder. For example, a patient may first be administered myricetin andsubsequently administered dexrazoxane, or may first be given dexraxozaneand then given myricetin.

In some cases, an anticancer agent (e.g., chemotherapeutic drug,biologic agent, protein kinase inhibitor, radiation therapy) (or othertreatment) and one or more protective agents of Formula 1 or Formula 2(e.g., myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, and myricitrin) may beadministered to a patient. In cases where a cancer treatment (or othertreatment) and at least two protective agents are administered to apatient, the cancer treatment (or other treatment) and the at least twoprotective agents (or derivative or salt thereof) may be administered asco-formulations in any combination. For example, a patient may beadministered a co-formulation of a protective agent and achemotherapeutic drug or a co-formulation containing one or morechemotherapeutic drugs and at least two protective agents.

In some cases, a patient or subject may be administered one or moreprotective agents (or derivative or salt thereof) and one or more cancertreatments (or other treatment) simultaneously. For example, the methodmay comprise administering to a patient a protective agent and achemotherapy as separate entities, but simultaneously.

In some cases, a patient or subject may be administered one or moreprotective agents of Formula 1 or Formula 2 (or derivative or saltthereof) and one or more cancer treatments (or other treatment)sequentially. For example, the protective agent may be administeredprior to administration of the cancer treatment (or other treatment).For example, a cancer patient may be administered a therapeuticallyeffective dose of myricetin to prevent cardiotoxicity, and subsequentlyadministered a chemotherapeutic drug (e.g., doxorubicin). In anotherexample, a cancer patient may be administered a therapeuticallyeffective dose of myricitrin to prevent cardiotoxicity, and subsequentlyadministered a chemotherapeutic drug (e.g., doxorubicin). In yet anotherexample, a cancer patient may be administered a therapeuticallyeffective dose of vitexin to prevent cardiotoxicity, and subsequentlyadministered a chemotherapeutic drug (e.g., doxorubicin). In anotherexample, a cancer patient may be administered a therapeuticallyeffective dose of robinetin to prevent cardiotoxicity, and subsequentlyadministered a chemotherapeutic drug (e.g., doxorubicin). In anotherexample, a cancer patient may be administered a therapeuticallyeffective dose of tricetin to prevent cardiotoxicity, and subsequentlyadministered a chemotherapeutic drug (e.g., doxorubicin). In otherexamples, the cancer treatment (or other treatment) is administered tothe patient or subject prior to administration of the protectiveagent(s) of Formula 1 or Formula 2. In some cases, the patient isadministered the one or more protective agents prior to receiving cancertreatment (or other treatment) and then is administered one or moreprotective agents following the cancer treatment.

In cases of sequential administration, there may be a delay periodbetween administration of the one or more protective agents and the oneor more cancer treatments (or other treatments). For example, theprotective agent may be administered minutes, hours, days, or weeksprior to administration of a cancer treatment or other treatment (e.g.,at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9hours, at least 10 hours, at least 1 day, at least 2 days, at least 3days, at least 5 days, at least 1 week, at least 2 weeks, at least 3weeks, at least 4 weeks, at least 2 months, at most 2 months, at most 1month, at most 3 weeks, at most 2 weeks, at most 1 week, at most 6 days,at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most1 day, at most 12 hours, at most 6 hours, at most 4 hours, at most 3hours, at most 2 hours or at most 1 hours prior to administration of thecancer treatment). In some cases, the protective agent has beenadministered to the patient at least 1 day prior to the cancertreatment. In some cases, the protective agent has been administered atmost 1 day prior to the cancer treatment. In some cases, the protectiveagent is administered at most within 2 hours after the cancer treatment.In some cases, the protective agent is administered at most within 4hours after the cancer treatment. In some cases, the protective agent isadministered at most within 6 hours after the cancer treatment. In somecases, the protective agent is administered at most within 12 hoursafter the cancer treatment. In some cases, the protective agent isadministered at most within 1 day after the cancer treatment. In somecases, the protective agent is administered at most within 2 days afterthe cancer treatment. In some cases, the protective agent isadministered at most within 3 days after the cancer treatment. In somecases, the protective agent is administered at most within 4 days afterthe cancer treatment. In some cases, the protective agent isadministered at most within 5 days after the cancer treatment.

The compounds of the current disclosure (e.g., the protective agents ofFormula 1) can be administered to a patient every time the patient isdosed with an anticancer agent with a dosage regimen described herein.For example, the protective agent may be administered to a patientwithin 24 hours every time before the patient is scheduled to be dosedwith an anticancer agent. In some cases, the protective agent can beadministered to a patient within 48 hours every time before the patientis scheduled to dosed with an anticancer agent. In some cases, theprotective agent can be administered concurrently to a patient everytime the patient is dosed with an anticancer agent. In some cases, theprotective agent can be administered to a patient every time the patienthas been dosed with an anticancer agent within at least 24 hoursfollowing the cancer treatment.

The compounds of the current disclosure may be administered by any ofthe accepted modes of administration of agents having similar utilities,for example, by cutaneous, oral, topical, intradermal, intrathecal,intravenous, subcutaneous, intramuscular, intra-articular, intraspinalor spinal, nasal, epidural, rectal, vaginal, or transdermal/transmucosalroutes. The most suitable route will depend on the nature and severityof the condition being treated. Subcutaneous, intradermal andpercutaneous injections can be routes for the compounds of thisdisclosure. Sublingual administration may be a route of administrationfor compounds of this disclosure. Intravenous administration may be aroute of administration for compounds of this disclosure. In aparticular example, the pharmaceutical composition provided herein maybe administered to a patient orally. In another particular example, thepharmaceutical composition comprising a protective agent provided hereinmay be administered to a patient intravenously (via, e.g., injection orinfusion). In another particular example, the pharmaceutical compositioncomprising a protective agent provided herein may be administered to apatient intramuscularly. In a particular example, the pharmaceuticalcomposition comprising a protective agent provided herein may beadministered to a patient nasally.

A pharmaceutical composition (e.g., for oral administration or forinjection, infusion, subcutaneous delivery, intramuscular delivery,intraperitoneal delivery, sublingual delivery, or other method) may bein the form of a liquid. A liquid pharmaceutical composition mayinclude, for example, one or more of the following: a sterile diluentsuch as water, saline solution, preferably physiological saline,Ringer's solution, isotonic sodium chloride, fixed oils that may serveas the solvent or suspending medium, polyethylene glycols, glycerin,propylene glycol or other solvents; antibacterial agents; antioxidants;chelating agents; buffers and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. The use of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile. In another embodiment,for treatment of an ophthalmological condition or disease, a liquidpharmaceutical composition may be applied to the eye in the form of eyedrops. A liquid pharmaceutical composition may be delivered orally.

For oral formulations, at least one of the compounds or agents describedherein can be used alone or in combination with appropriate additives tomake tablets, powders, granules or capsules, and if desired, withdiluents, buffering agents, moistening agents, preservatives, coloringagents, and flavoring agents. The compounds may be formulated with abuffering agent to provide for protection of the compound from low pH ofthe gastric environment and/or an enteric coating. A compound includedin a pharmaceutical composition may be formulated for oral delivery witha flavoring agent, e.g., in a liquid, solid or semi-solid formulationand/or with an enteric coating. In some cases, the compounds of thisdisclosure may be solubilized and encapsulated (e.g., in a liposome or abiodegradable polymer), or used in the form of microcrystals coated withan appropriate nontoxic lipid. In some cases, the compounds of thisdisclosure may be solubilized and encapsulated in a liposome, micelle orthe both.

A pharmaceutical composition comprising any one of the compounds oragents described herein may be formulated for sustained or slow release(also called timed release or controlled release). Such compositions maygenerally be prepared using well known technology and administered by,for example, oral, rectal, intradermal, or subcutaneous implantation, orby implantation at the desired target site. Sustained-releaseformulations may contain the compound dispersed in a carrier matrixand/or contained within a reservoir surrounded by a rate controllingmembrane. Excipients for use within such formulations are biocompatible,and may also be biodegradable; preferably the formulation provides arelatively constant level of active component release. Non-limitingexamples of excipients include water, alcohol, glycerol, chitosan,alginate, chondroitin, Vitamin E, mineral oil, and dimethyl sulfoxide(DMSO). The amount of compound contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release, and the nature of the condition, disease ordisorder to be treated or prevented.

The disclosure provided herein also describes methods for preventing,reducing, or eliminating organ damage in a subject by administering to apatient one or more protective agents of Formula 1 or Formula 2. Theprotective agent of Formula 1 or Formula 2 for preventing, reducing, oreliminating organ damage in a subject can include without limitationmyricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, or myricitrin (orderivative or salt thereof). In particular, the organ damage may becaused by cardiac tissue damage, electrophysiological dysfunction,mitochondrial toxicity, apoptosis, or oxidative stress, leading to heartfailure. For example, a pharmaceutical composition comprising a compoundof Formula 1 (i.e., protective agent) may be administered to a patientthat is experiencing cancer treatment-induced heart failure, whereinfurther hear failure is prevented by the administration of thepharmaceutical composition.

The pharmaceutical methods and compositions described herein prevent,reduce, or eliminate cancer treatment-induced cardiotoxicity in apatient. Accordingly, the methods and compositions provided hereinenable a patient (e.g., a heart-healthy patient, a patient with cardiacdisease) to receive a higher dosage of a therapy without having thedosage regimen significantly altered by the risk of cardiotoxicity. Insome cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of greater than 0.1mg/m², 1 mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8mg/m², 9 mg/m², 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15mg/m², 16 mg/m², 17 mg/m², 18 mg/m², 19 mg/m², 20 mg/m², 21 mg/m², 22mg/m², 23 mg/m², 24 mg/m², 25 mg/m², 26 mg/m², 27 mg/m², 28 mg/m², 29mg/m², 30 mg/m², 31 mg/m², 32 mg/m², 33 mg/m², 34 mg/m², 35 mg/m², 36mg/m², 37 mg/m², 38 mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43mg/m², 44 mg/m², 45 mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50mg/m², 100 mg/m², 150 mg/m², 200 mg/m², 300 mg/m², 350 mg/m², 400 mg/m²,450 mg/m², 500 mg/m², 750 mg/m², 1000 mg/m², 1250 mg/m², 1500 mg/m²,1750 mg/m², or 2000 mg/m² of chemotherapeutic drug (e.g., anthracycline,doxorubicin or derivative or salt thereof) to a patient.

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of about 0.1 mg/m², 0.2mg/m², 0.3 mg/m², 0.4 mg/m², 0.5 mg/m², 0.6 mg/m², 0.7 mg/m², 0.8 mg/m²,0.9 mg/m², 1 mg/m², 1.1 mg/m², 1.2 mg/m², 1.3 mg/m², 1.4 mg/m², 1.5mg/m², 1.6 mg/m², 1.7 mg/m², 1.8 mg/m², 1.9 mg/m², 2 mg/m², 2.1 mg/m²,2.2 mg/m², 2.3 mg/m², 2.4 mg/m², 2.5 mg/m², 2.6 mg/m², 2.7 mg/m², 2.8mg/m², 2.9 mg/m², 3 mg/m², 3.1 mg/m², 3.2 mg/m², 3.3 mg/m², 3.4 mg/m²,3.5 mg/m², 3.6 mg/m², 3.7 mg/m², 3.8 mg/m², 3.9 mg/m², 4 mg/m², 4.1mg/m², 4.2 mg/m², 4.3 mg/m², 4.4 mg/m², 4.5 mg/m², 4.6 mg/m², 4.7 mg/m²,4.8 mg/m², 4.9 mg/m², 5 mg/m², 5.1 mg/m², 5.2 mg/m², 5.3 mg/m², 5.4mg/m², 5.5 mg/m², 5.6 mg/m², 5.7 mg/m², 5.8 mg/m², 5.9 mg/m², 6 mg/m²,6.1 mg/m², 6.2 mg/m², 6.3 mg/m², 6.4 mg/m², 6.5 mg/m², 6.6 mg/m², 6.7mg/m², 6.8 mg/m², 6.9 mg/m², 7 mg/m², 7.1 mg/m², 7.2 mg/m², 7.3 mg/m²,7.4 mg/m², 7.5 mg/m², 7.6 mg/m², 7.7 mg/m², 7.8 mg/m², 7.9 mg/m², 8mg/m², 8.1 mg/m², 8.2 mg/m², 8.3 mg/m², 8.4 mg/m², 8.5 mg/m², 8.6 mg/m²,8.7 mg/m², 8.8 mg/m², 8.9 mg/m², 9 mg/m², 9.1 mg/m², 9.2 mg/m², 9.3mg/m², 9.4 mg/m², 9.5 mg/m², 9.6 mg/m², 9.7 mg/m², 9.8 mg/m², 9.9 mg/m²,10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16 mg/m², 17mg/m², 18 mg/m², 19 mg/m², 20 mg/m², 21 mg/m², 22 mg/m², 23 mg/m², 24mg/m², 25 mg/m², 26 mg/m², 27 mg/m², 28 mg/m², 29 mg/m², 30 mg/m², 31mg/m², 32 mg/m², 33 mg/m², 34 mg/m², 35 mg/m², 36 mg/m², 37 mg/m², 38mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43 mg/m², 44 mg/m², 45mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50 mg/m², 51 mg/m², 52mg/m², 53 mg/m², 54 mg/m², 55 mg/m², 56 mg/m², 57 mg/m², 58 mg/m², 59mg/m², 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65 mg/m², 66mg/m², 67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m², 72 mg/m², 73mg/m², 74 mg/m², 75 mg/m², 76 mg/m, 77 mg/m², 78 mg/m, 79 mg/m², 80mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m², 85 mg/m², 86 mg/m², 87mg/m², 88 mg/m², 89 mg/m², 90 mg/m², 91 mg/m², 92 mg/m², 93 mg/m², 94mg/m², 95 mg/m², 96 mg/m², 97 mg/m², 98 mg/m², 99 mg/m², or 100 mg/m² ofa biologic agent to a patient.

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of about 0.1 mg/m², 1mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8 mg/m², 9mg/m², 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16mg/m², 17 mg/m², 18 mg/m², 19 mg/m², 20 mg/m², 21 mg/m², 22 mg/m², 23mg/m², 24 mg/m², 25 mg/m², 26 mg/m², 27 mg/m², 28 mg/m², 29 mg/m², 30mg/m², 31 mg/m², 32 mg/m², 33 mg/m², 34 mg/m², 35 mg/m², 36 mg/m², 37mg/m², 38 mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43 mg/m², 44mg/m², 45 mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50 mg/m², 51mg/m², 52 mg/m², 53 mg/m², 54 mg/m², 55 mg/m², 56 mg/m², 57 mg/m², 58mg/m², 59 mg/m², 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65mg/m², 66 mg/m², 67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m², 72mg/m², 73 mg/m², 74 mg/m², 75 mg/m², 76 mg/m², 77 mg/m², 78 mg/m², 79mg/m², 80 mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m², 85 mg/m², 86mg/m², 87 mg/m², 88 mg/m², 89 mg/m², 90 mg/m², 90 mg/m², 95 mg/m², 100mg/m², 110 mg/m², 120 mg/m², 130 mg/m², 140 mg/m², 150 mg/m², 160 mg/m²,170 mg/m², 180 mg/m², 190 mg/m², 200 mg/m², 300 mg/m², 400 mg/m², 500mg/m² of the protective agent drug of Formula 1 or Formula 2 (e.g.,myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrin and/or aderivative or salt thereof).

The daily fixed dose of protective agent described herein, or collectivedose of a combination of protective agents can be greater than 0.1 mg, 1mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg,13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 150 mg, 200mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 750 mg or higher of theprotective agent (or any derivative or salt thereof). In some cases, theprotective agent or agents is selected from the group consisting ofmyricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrin and/or aderivative or salt thereof. In a particular example, administering apharmaceutical composition to a patient can comprise administering aco-formulation of a chemotherapy drug (e.g., doxorubicin) with at least10 mg of myricetin. In some cases, administering a pharmaceuticalcomposition herein to a patient can comprise administering a daily doseof 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49mg, 50 mg, 100 mg, 500 mg, 750 mg, or 1 g of myricetin (or anyderivative or salt thereof) to a patient.

In another example, administering a pharmaceutical composition to apatient can comprise administering a co-formulation of a chemotherapydrug (e.g., doxorubicin) with at least 10 mg of myricetrin. In somecases, administering a pharmaceutical composition herein to a patientcan comprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3 mg, 0.4mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13 mg,14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750 mg, or1 g of myricetrin (or any derivative or salt thereof) to a patient.

In yet another example, administering a pharmaceutical composition to apatient can comprise administering a co-formulation of a chemotherapydrug (e.g., doxorubicin) with at least 10 mg of vitexin. In some cases,administering a pharmaceutical composition herein to a patient cancomprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg,0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg,1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg,2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.1 mg,3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4 mg,4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg,5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg,5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg,6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg,7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg,8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg,9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750 mg, or1 g of vitexin (or any derivative or salt thereof) to a patient.

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9mg, 4 mg, 4.1 mg, 4.2 mg, 43 mg, 44 mg, 4.5 mg, 46 mg, 4.7 mg, 4.8 mg,4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg,5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg,6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg,7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg,8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg,9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750mg, or 1 g of robinetin (or any derivative or salt thereof).

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6mg, 6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg,13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750mg, or 1 g of tricetin (or any derivative or salt thereof).

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9mg, 4 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8mg, 4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7mg, 5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 66 mg,67 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 75 mg, 7.6mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13 mg,14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750 mg, or1 g of 7,3′,4′,5′-tetrahydroxyflavone (or any derivative or saltthereof).

In some cases, administering a pharmaceutical composition herein to apatient can comprise administering a daily dose of 0.1 mg, 0.2 mg, 0.3mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.1mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9mg, 4 mg 4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg,4.9 mg, 5 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg,5.8 mg, 5.9 mg, 6 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg,6.7 mg, 6.8 mg, 6.9 mg, 7 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg,7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg,8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 9.1 mg, 9.2 mg, 9.3 mg,9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10 mg, 11 mg, 12 mg, 13mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 100 mg, 500 mg, 750mg, or 1 g of ficetin (or any derivative or salt thereof).

The pharmaceutical methods and compositions described herein prevent,reduce, or eliminate cancer treatment-induced cardiotoxicity in apatient. Accordingly, the methods and compositions provided hereinenable a patient to receive a therapy more frequently without having thedosage regimen significantly altered by the risk of cardiotoxicity. Thedaily dose of a chemotherapeutic drug, biologic agent or protectiveagent within the pharmaceutical composition provided herein may beadministered to a patient in one or more doses per day. In some cases,the daily dose of the chemotherapeutic drug may be administered in asingle dose. In some cases, the daily dose of the chemotherapeutic drugmay be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses per day. Forexample, the daily dose of chemotherapeutic drug (e.g., doxorubicin) canbe divided into 3 doses per day. In some cases, the daily dose of thechemotherapeutic drug may be divided into at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 infusions per hour.In some cases, each infusion of a composition comprising achemotherapeutic drug may last for at least 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45minutes, 50 minutes, 55 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours,3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours.In some cases, the daily dose of the biologic agent may be administeredin a single dose. In some cases, the daily dose of the biologic agentmay be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 doses per day. For example, thedaily dose of biologic agent (e.g., bevacizumab) can be divided into 3doses per day. In some cases, the daily dose of the biologic agent maybe divided into at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, or 60 infusions per hour. In some cases, eachinfusion of a composition comprising a biologic agent may last for atleast 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5hours, 5 hours, 5.5 hours, or 6 hours. In some cases, the daily dose ofthe protective agent may be administered in a single dose. In somecases, the daily dose of the protective agent may be divided into 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 doses per day. For example, the daily dose of protective agent(e.g., myricetin) can be divided into 3 doses per day. In some cases,the daily dose of the protective agent may be divided into at least 1,2, 3, 4, 5, or 6 infusions per hour. In some cases, each infusion of acomposition comprising one or more protective agent(s) may last for atleast 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5hours, 5 hours, 5.5 hours, or 6 hours.

The pharmaceutical compositions described herein may be administered toa patient one or more times per day. In some cases, the pharmaceuticalcomposition may be administered to a patient one time per day. In somecases, the pharmaceutical composition may be administered to a patientat least 2 times, 3 times, 4 times 5 times, 6 times, 7 times, 8 times, 9times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23times, or 24 times per day. For example, a pharmaceutical compositionmay be administered to a patient 3 times per day.

The pharmaceutical compositions described herein may be administered toa patient for one or more days. In some cases, the pharmaceuticalcomposition may be administered to a patient for one day. In some cases,the pharmaceutical composition may be administered to the patient for atleast 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks,1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 20 years,30 years, 40 years, or 50 years. For example, a cancer patient may beadministered a pharmaceutical co-formulation of doxorubicin andmyricetin for a period of at least 1 year. In some cases, thepharmaceutical composition may be administered to a patient for two ormore consecutive days. In some cases, the pharmaceutical composition maybe administered to a patient for two or more non-consecutive days. Forexample, a patient may be administered a pharmaceutical compositionevery day, consecutively, for 4 days. In another example, a patient maybe administered a pharmaceutical composition on day 1, day 3, day 7, andday 15. In some cases, when a patient is administered a pharmaceuticalcomposition over a period of time, the dosage amount administered to thepatient on one day can be different from the dosage amount administeredto the patient on a subsequent day. For example, a patient may beadministered 5 mg of a pharmaceutical composition on the first day, andadministered 10 mg of a pharmaceutical composition on a subsequent day.

The pharmaceutical compositions described herein may be effective overtime. In some cases, the pharmaceutical composition may be effective forone or more days. In some cases, the duration of efficacy of thepharmaceutical composition is over a long period of time. In some cases,the efficacy of the pharmaceutical composition may be greater than 2days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1month.

Methods provided herein can further comprise administering to thepatient dexrazoxane (or any derivative or salt thereof) as part of anyof the pharmaceutical compositions described herein. Such methods allowfor the administration to a patient a pharmaceutical compositioncontaining at least one protective agent and dexrazoxane, wherein theco-formulation of at least one protective agent and dexrazoxane canprovide a greater protective effect as compared to the administration ofdexrazoxane alone. In some cases, the administration of any of thepharmaceutical compositions described herein can reduce the likelihoodof cardiotoxicity across a patient pool by as much as 1%, 2%, 3%, 4%,5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, For example, ifthere is an 80% likelihood that patients in a patient pool that areadministered dexrazoxane will experience cardiotoxicity, administeringto the patients a co-formulation of myricetin and dexrazoxane can reducethe likelihood of experiencing cardiotoxicity by 75%, resulting in a 20%likelihood that the patients will experience cardiotoxicity. Thisgreater protective effect may also enable a larger population ofpatients, including those with pre-existing cardiac conditions, toreceive a cancer treatment (e.g., doxorubicin) to which they wouldotherwise be precluded. In some cases, the dexrazoxane may beco-formulated within the pharmaceutical composition, in that it is mixedwithin the pharmaceutical composition, or exist as a distinct entity. Insome cases, the cancer treatment, protective agent, and dexrazoxane maybe administered concurrently. In some cases, the cancer treatment,protective agent, and dexrazoxane may be administered sequentially. Inone example, a cancer patient may be administered a co-formulation ofchemotherapeutic drug, dexrazoxane, and myricetin in a single dose atleast one time per day. In another example, a cancer patient may beadministered dexrazoxane, and subsequently administered myricetin.

The dose of dexrazoxane (or any derivative or salt thereof) administeredwithin the pharmaceutical composition can be greater than 0.1 mg/m², 1mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8 mg/m², 9mg/m², 10 mg/m², 11 mg/m², 12 mg/m², 13 mg/m², 14 mg/m², 15 mg/m², 16mg/m², 17 mg/m², 18 mg/m², 19 mg/m², 20 mg/m², 21 mg/m², 22 mg/m², 23mg/m², 24 mg/m², 25 mg/m², 26 mg/m², 27 mg/m², 28 mg/m², 29 mg/m², 30mg/m², 31 mg/m², 32 mg/m², 33 mg/m², 34 mg/m², 35 mg/m², 36 mg/m², 37mg/m², 38 mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43 mg/m², 44mg/m², 45 mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50 mg/m², 51mg/m², 52 mg/m², 53 mg/m², 54 mg/m², 55 mg/m², 56 mg/m², 57 mg/m², 58mg/m², 59 mg/m², 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65mg/m², 66 mg/m², 67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m², 72mg/m², 73 mg/m², 74 mg/m², 75 mg/m², 76 mg/m², 77 mg/m², 78 mg/m², 79mg/m², 80 mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m², 85 mg/m², 86mg/m², 87 mg/m², 88 mg/m², 89 mg/m², 90 mg/m², 91 mg/m², 92 mg/m², 93mg/m², 94 mg/m², 95 mg/m², 96 mg/m², 97 mg/m², 98 mg/m², 99 mg/m², 100mg/m², 150 mg/m², 200 mg/m², 300 mg/m², 350 mg/m², 400 mg/m², 450 mg/m²,500 mg/m², 750 mg/m², 1 g/m², 5 g/m², 10 g/m², or higher. In aparticular example, administering a pharmaceutical composition to apatient can comprise administering a co-formulation of a protectiveagent of Formula 1 or Formula 2 (e.g., myricetin) with 50 mg/m² ofdexrazoxane.

In some cases, administering a pharmaceutical composition describedherein to a patient can comprise administering a dose of about 0.1mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg,0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg,3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, 50 mg/kg, 51mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57 mg/kg, 58mg/kg, 59 mg/kg, 60 mg/kg, 61 mg/kg, 62 mg/kg, 63 mg/kg, 64 mg/kg, 65mg/kg, 66 mg/kg, 67 mg/kg, 68 mg/kg, 69 mg/kg, 70 mg/kg, 71 mg/kg, 72mg/kg, 73 mg/kg, 74 mg/kg, 75 mg/kg, 76 mg/kg, 77 mg/kg, 78 mg/kg, 79mg/kg, 80 mg/kg, 81 mg/kg, 82 mg/kg, 83 mg/kg, 84 mg/kg, 85 mg/kg, 86mg/kg, 87 mg/kg, 88 mg/kg, 89 mg/kg, 90 mg/kg, 90 mg/kg, 95 mg/kg, 100mg/kg, 110 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg,170 mg/kg, 180 mg/kg, 190 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500mg/kg of a protective agent of Formula 1 or Formula 2. In some aspects,the protective agent can be selected from the group consisting ofmyricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrin and aderivative or salt thereof. In one embodiment, the patient isadministered intravenously with a protective agent at 0.5 mg/kg, 1mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 mg/kg,12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47mg/kg, 48 mg/kg, 49 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90mg/kg, or 100 mg/kg, 150 mg/kg, or 200 mg/kg. In one embodiment, thepatient is administered with myricetin at a dose between about 0.5 mg/kgand about 50 mg/kg at least 10 minutes before administering ananthracycline (e.g., doxorubicin, epirubicin, or idarubicin). In oneembodiment, the patient is administered with myricetin at a dose betweenabout 0.5 mg/k and about 100 mg/kg at least 10 minutes beforeadministering an anthracycline (e.g., doxorubicin, epirubicin, oridarubicin). In one embodiment, the patient is administeredintravenously with myricetin at a dose between about 0.5 mg/kg and about200 mg/kg at least 30 minutes prior to the administration of ananthracycline (e.g., doxorubicin, epirubicin, or idarubicin). In oneembodiment, the patient is administered intravenously with myricetin ata dose between about 0.5 mg/kg and about 200 mg/kg at least 1 hour priorto the administration of an anthracycline (e.g., doxorubicin,epirubicin, or idarubicin). In one embodiment, the patient isadministered intravenously with myricetin at a dose between about 0.5mg/kg and about 200 mg/kg of myricetin at least 2 hours before theadministration of an anthracycline (e.g., doxorubicin, epirubicin, oridarubicin). In one embodiment, the patient is administeredintravenously with a dose between about 0.5 mg/kg and about 200 mg/kg ofmyricetin at least 4 hours prior to the administration of ananthracycline (e.g., doxorubicin, epirubicin, or idarubicin). In oneembodiment, the patient is administered intravenously with myricetin ata dose between about 0.5 mg/kg and about 200 mg/kg of myricetin at least6 hours before the administration of an anthracycline (e.g.,doxorubicin, epirubicin, or idarubicin). In one embodiment, the patientis administered intravenously with myricetin at a dose between about 0.5mg/kg and about 200 mg/kg of myricetin within 6 hours after theadministration of an anthracycline (e.g., doxorubicin, epirubicin, oridarubicin). In one embodiment, myricetin is administered orally at adose between 0.5 mg/kg and 200 mg/kg at least 0.5, 1, 2, 3, 4, 5, or 6hour(s) prior to the administration of an anthracycline (e.g.,doxorubicin, epirubicin, or idarubicin).

In some aspects, the patient is administered, for example, intravenouslyor orally with a protective agent (e.g., myricetin, vitexin, robinetin,tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, ormyrcitrin) at a dose between about 0.5 mg/kg and about 200 mg/kg atleast 4 hours prior to the first administration of an anthracycline(e.g., doxorubicin, epirubicin, or idarubicin) after the patient hasbeen diagnosed with cancer.

Patient Response

The methods and compositions provided herein prevent, reduce, oreliminate cardiotoxicity in a patient caused by chemotherapeutic drugs,biologic agents, or radiation therapy. Furthermore, administering to apatient a pharmaceutical composition disclosed herein may also prevent,reduce or eliminate cancer treatment-induced organ damage (e.g., organdamage caused by cardiac tissue damage, electrophysiologicaldysfunction, mitochondrial toxicity, apoptosis, or oxidative stress).

The methods and compositions disclosed herein may generally reducecardiotoxicity in a patient. Examples of cardiotoxicity can include, butare not limited to, mitochondrial toxicity, apoptosis,electrophysiological dysfunction (e.g., QT prolongation), mechanicaldysfunction (e.g., reduced cardiac ejection fraction), oxidative stress,cardiac tissue damage (e.g., damage caused by oxidative stress,mitochondrial damage, or damage caused by an increase in the flux ofreactive oxygen species), and cytotoxic injury to any organ (e.g.,liver, kidney, or pancreas) that is not the heart.

Mitochondrial toxicity can refer to any damage that decreases the numberof the active mitochondria within a given cell, tissue, organ, ororganism. In some cases, mitochondrial toxicity can be measured using anin vitro assay. One such method that can be used for measuringmitochondrial toxicity is by co-exposing cells to (1) a cell-permeablefluorescent dye that indicates cellular nuclei, and (2)tetramethylrhodamine methyl ester (TMRM), a cell-permeable fluorescentdye that is sequestered by active mitochondria. Mitochondrial toxicitycan be calculated as the fraction of TMRM-positive cells to the totalnumber of cell nuclei. As measured by the in vitro assay, cancertreatment-induced mitochondrial toxicity may be greater than 1%, 2%, 3%,4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in cardiomyocytes,as compared to untreated controls. For example, exposing cardiomyocytesto 1 micromolar of doxorubicin for at least 48 hours can cause 100%mitochondrial toxicity, as compared to untreated control. Thepharmaceutical methods and compositions described herein generallyreduce cancer treatment-induced mitochondrial toxicity. As measured bythe in vitro assay, exposing cardiomyocytes to any of the pharmaceuticalcompositions described herein can reduce mitochondrial toxicity as muchas 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%,73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%,59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%,45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%,31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% as compared to cardiomyocytes exposed to a cancer treatment in theabsence of a protective agent. For example, exposing cardiomyocytes to aco-formulation of 1 micromolar doxorubicin and 115 micromolar ofmyricetin for at least 48 hours can reduce mitochondrial toxicity by30%, as compared to cardiomyocytes exposed to 1 micromolar ofdoxorubicin.

Apoptosis can refer to a process by which a cell undergoes programmedcell death. Detectable changes within a cell undergoing apoptosisinclude, but are not limited to, the translocation of cytochrome C fromthe mitochondria, diminished mitochondrial function, changes in membranestructure, increased proteolytic activity, and DNA fragmentation. Insome cases, apoptosis can be measured using an in vitro assay. One suchmethod that can be used for measuring apoptosis is by co-exposing cellsto (1) a cell-permeable fluorescent dye that indicates cellular nuclei,and (2) CellEvent Caspase 3/7 Detection Reagent, a fluorogenic substratefor the activated caspase 3 that is uniquely present in apoptotic cells.Percentage apoptosis can be calculated as the fraction ofCellEvent-positive cells to the total number of cell nuclei. As measuredby the in vitro assay, cancer treatment-induced apoptosis may be greaterthan 1%, 2%, 3%, 4%, 5%, 6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% incardiomyocytes, as compared to untreated controls. For example, exposingcardiomyocytes to 1 micromolar of doxorubicin for at least 48 hours cancause 100% apoptosis, as compared to untreated control.

The pharmaceutical methods and compositions described herein maygenerally reduce cancer treatment-induced apoptosis. As measured by thein vitro assay, exposing cardiomyocytes to any of the pharmaceuticalcompositions described herein can reduce apoptosis as much as 100%, 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%,56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%,42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%,28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% ascompared to cardiomyocytes exposed to a cancer treatment in the absenceof a protective agent. For example, exposing cardiomyocytes to aco-formulation of 1 micromolar doxorubicin and 115 micromolar ofmyricetin for at least 48 hours can reduce mitochondrial toxicity by30%, as compared to cardiomyocytes exposed to 1 micromolar doxorubicin.

Electrophysiological dysfunction can refer to any damage wherein theflow of ions through a biological tissue is disrupted. For example,administering to a cancer patient a chemotherapeutic drug (e.g.,doxorubicin) may cause an acute myocardial infarction, wherein ions canno longer flow through the damaged cardiac tissue resulting in aconduction block. In some cases, electrophysiological dysfunction cancomprise prolongation of the QT interval, and can be measured using anin vivo assay. The QT interval can be used to describe the time betweenthe start of the Q wave and the end of the T wave in anelectrocardiogram. QT prolongation may indicate delayed ventricularrepolarization, and can predispose the heart to earlyafter-depolarizations (EADs) leading to re-entrant arrhythmia (e.g.,Torsades de Pointes). A QT interval may also depend on the length of thecardiac cycle (RR), the amount of time between the onset of one QRScomplex and the onset of the next QRS complex. A corrected QT (QTc)interval may be used to represent a QT interval that has been correctedto account for the cycle length. Bazett's formula (QTc=QT/√RR),Fridericia's formula (QTc=QT/³√RR), or a regression analysis method(QTc=QT+0.154(1−RR)) may all be used to calculate the QTc interval fromthe QT interval.

Administration of a chemotherapeutic drug, biologic agent, or radiationtherapy to a patient may cause QTc prolongation, above the baseline QTcinterval of the patient, in the absence of a protective agent. Thebaseline QTc interval for a patient is the QTc interval measured in thepatient prior to the administration of any drug. For example,administering to a patient a chemotherapeutic drug (e.g., doxorubicin)in the absence of a protective agent can cause a QTc prolongation of 40milliseconds (ms) above the baseline QTc interval for the patient. Insome cases, administration of a chemotherapeutic drug, biologic agent,or radiation therapy to a patient, particularly in the absence of aprotective agent, may cause a QTc prolongation of at least 1 ms, 2 ms, 3ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 15 ms, 20 ms, 25 ms, 30ms, 35 ms, 40 ms, 45 ms, 50 ms, 55 ms, 60 ms, 65 ms, 70 ms, 75 ms, 80ms, 85 ms, 90 ins, 95 ms, or 100 ms above the baseline QTc interval ofthe patient.

Administration of any of the pharmaceutical compositions describedherein can limit the cancer treatment-induced QTc prolongationexperienced by the patient, above the baseline QTc interval of thepatient. For example, administering to a patient a co-formulation of achemotherapeutic drug (e.g., doxorubicin) and a protective agent (e.g.,myricetin) can cause a QTC prolongation of less than 5 ms. In somecases, the pharmaceutical compositions described herein can cause lessthan a 100 ms, 95 ms, 90 ms, 85 ms, 80 ms, 75 ms, 70 ms, 65 ms, 60 ms,55 ms, 50 ms, 45 ms, 40 ms, 35 ms, 30 ms, 25 ms, 20 ms, 15 ms, 10 ms, 9ms, 8 ms, 7 ms, 6 ms, 5 ms, 4 ms, 3 ms, 2 ms, or 1 ms increase in QTcprolongation above the baseline QTc interval of the patient.

In some cases, electrophysiological dysfunction can also comprisediminished electrical activity, and can be measured using an in vitroassay. Multielectrode arrays (MEAs) are devices that contain multipleplanar conductive electrodes on which cells (e.g., cardiomyocytes) maybe contacted. Although the size and shape of the electrical recordingmeasured from an MEA can depend on several factors (e.g., cellhomogeneity, contact between the cell and an electrode, geometry of anMEA), temporal changes can be measured by the electrode to provideinformation on the electrical activity of the contacting cells (e.g.,percentage of active electrodes, field potential duration, and beatrate).

Exposing cardiomyocytes to a chemotherapeutic drug, biologic agent, orradiation therapy, in the absence of a protective agent, may cause atemporal decrease in the percentage of active electrodes (e.g., anelectrode that is able to measure some electrical activity from thecontacting cell), as measured by the in vitro assay. For example,exposing cardiomyocytes to 1 micromolar of doxorubicin for at least 24hours can cause a 50% decrease in the number of active electrodes, ascompared to time zero. In some cases, exposing cardiomyocytes to acancer treatment (e.g., doxorubicin) in the absence of a protectiveagent (e.g., myricetin) can cause as much as a 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,810%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%,67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%,53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%,39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%,25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% reduction in the numberof active electrodes, as measured by the in vitro assay. In a particularexample, exposing cardiomyocytes to 1 μM doxorubicin for at least 24hours can cause as much as a 50% reduction in the number of activeelectrodes.

The pharmaceutical methods and compositions described herein generallyreduce cancer treatment-induced electrophysiological dysfunction (e.g.,decrease in the number of active electrodes). As measured by the invitro assay, exposing cardiomyocytes to any of the pharmaceuticalcompositions described herein may induce less than a 1%, 2%, 3%, 4%, 5%,6%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% decrease in the number ofactive electrodes, as compared to cardiomyocytes exposed to a cancertreatment in the absence of a protective agent. For example, exposingcardiomyocytes to a co-formulation of 1 μM doxorubicin and 100 μMmyricetin for at least 24 hours can induce less than a 5% decrease inthe number of active electrodes.

The pharmaceutical methods and compositions described herein generallyreduce the risk that the patient will experience cardiotoxicity with theadministration of a cancer treatment. In some cases, the pharmaceuticalmethods and compositions described herein can reduce the risk ofcardiotoxicity in the patient by 100%, 99%, 98%, 97%, 96%, 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%,79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%,51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%,37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 80, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, In some cases, the pharmaceuticalmethods and compositions disclosed herein may reduce the risk ofcardiotoxicity in the patient by greater than 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%, For example, if a patient has a 90% risk for experiencing QTprolongation when administered a chemotherapeutic drug (e.g.,doxorubicin, epirubicin, or idarubicin) in the absence of a protectiveagent (e.g., myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, myricitrin and/or a derivative or saltthereof), the patient may experience a 50% reduction of risk for QTprolongation when the protective agent is administered separately or asa co-formulation with a chemotherapeutic drug, resulting in a 45% riskfor QT prolongation in the patient. For example, in one particularlyembodiment, the patient is administered intravenously with a protectiveagent (e.g., myricetin, vitexin, robinetin, tricetin, ficetin,7,3′,4′,5′-tetrahydroxyflavone, myricitrin and/or a derivative or saltthereof) at a dose between about 0.5 mg/kg and about 100 mg/kg at least30 minute, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours priorto the administration of chemotherapeutic drug (e.g., doxorubicin,epirubicin, or idarubicin), wherein the risk for QT prolongation isreduced by at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared tothat of control that did not receive the protective agent.

The effect of anthracycline-induced cardiotoxicity on contractility canbe also assessed by measuring fractional shortening (FS) and ejectionfraction (EF) which are indices of systolic function. An anthracyclinesuch as doxorubicin can have a profound impact on contractileproperties. However, a patient administered with a protective agent ofFormula 1 or Formula 2 (e.g., myricetin, vitexin, robinetin, tricetin,ficetin, 7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin, myricitrinand/or a derivative or salt thereof) can experience significantlyreduced, e.g., doxorubicin-induced cardiotoxicity as observed by markedimprovements in FS and EF. For example, myricetin can rescueanthracycline-induced FS and EF dysfunction by at least 30%, 40%, 50%,60%, 70%, 80%, 90% in the patient as compared to a control group thathas been treated with anthracycline, but not dosed with the protectiveagent.

The term “about” when referring to a number or a numerical range meansthat the number or numerical range referred to is an approximationwithin experimental variability (or within statistical experimentalerror), and thus the number or numerical range may vary from, forexample, between 1% and 10% of the stated number or numerical range.

The term “therapeutically effective amount” may generally refer to theamount (or dose) of a compound or other therapy that is minimallysufficient to prevent, reduce, treat or eliminate a condition, or riskthereof, when administered to a subject in need of such compound orother therapy. In some instances, the term “therapeutically effectiveamount” may refer to that amount of compound or other therapy that issufficient to have a prophylactic effect when administered to a subject.The therapeutically effective amount may vary; for example, it may varydepending upon the subject's condition, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, all of which may be determined by one ofordinary skill in the art.

Compositions

The pharmaceutical compositions disclosed herein may comprise aprotective agent disclosed in Formula 1 or Formula 2 (e.g., myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,dihydrorobinetin, myricitrin and/or a derivative or salt thereof). Thepharmaceutical composition may comprise one or more protective agents inany combination, two or more agents in any combination, three or moreprotective agents in any combination, or four or more protective agentsin any combination. In some cases, the pharmaceutical composition can bea co-formulation of at least two protective agents (e.g., myricetin,vitexin, robinetin, tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone,myricetin (myricitrin), dexrazoxane, and/or a derivative or saltthereof), or a co-formulation of at least one protective agent and acancer treatment (e.g., chemotherapeutic drug, biologic agent, proteinkinase inhibitor or radiation therapy). The protective agents within thepharmaceutical composition may reduce, eliminate or preventcardiotoxicity induced by the cancer treatment. Additionally, theprotective agents within the pharmaceutical composition may also reduce,eliminate or prevent organ damage induced by the cancer treatment. Inone example, this disclosure provides a co-formulation comprisingmyricetin and dexrazoxane. In another example, this disclosure providesa co-formulation comprising the chemotherapeutic drug doxorubicin andmyricetin.

In some cases, at least one of the protective agents in the compositionmay be a flavonoid, or a derivative thereof. Generally, a flavonoid maybe any compound with a 15-carbon skeleton backbone consisting of twophenyl and one heterocyclic ring. Flavonoids may belong to any of thefollowing classes of compounds including, but not limited to,anthroxanthins, flavanones, flavonols, flavanonols, flavans,anthocyanadins, bioflavonoids, isoflavonoids, isoflavones, isofiavanes,isoflavandiols, isoflavenes, or neoflavonoids. Non-limiting examples offlavonoids include ayanin, carlinoside, dihydrodaidzein,dihydroobavatin, irigenin, isoanhydroicaritin, isokurarinone,isoxanthohumol, gardenin, lupiwighteone, methoxypuerarin, mirificin,myricetin, myricetrin (myricitrin), dihydromyricetin, pyrroside,kaempferol, quercetin, swertisin, syzalterin, tricetin, ficetin,robinetin, dihydrorobinetin, 7,3′,4′,5′-tetrahydoxylflavone,5,7,3′,4′,5′-pentahydoxyflavone or thevetiaflavone. In one example, apharmaceutical composition disclosed herein may comprise the flavonesuch as 7,3′,4′,5′-tetrahyodxyflavone and tricetin. In another example,a pharmaceutical composition disclosed herein may comprise the flavonolsuch as myricetin, ficetin, robinetin, quercetin and kaempferol. Inanother example, a pharmaceutical composition disclosed herein maycomprise myricetrin. In an additional example, a pharmaceuticalcomposition disclosed herein may comprise the flavanolol such asdihydromyricetin and dihydrorobinetin. In yet another example, apharmaceutical composition disclosed herein may comprise aco-formulation of dexrazoxane and the flavonoid myricetin. Inparticular, the flavonoid myricetin may regulate mitochondrial toxicityin the heart by altering the activity of pyruvate dehydrogenase kinase(PDK4), a protein that may regulate enzymatic activity in cardiac tissue

In some cases, the pharmaceutical compositions described herein maycomprise a compound according to Formula 1,

wherein:

X¹ is CR⁵R⁶, NR⁵, O, S, C═O, or C═S;

each of R¹, R², R³, R⁵, R⁶, R⁹, and R¹⁰ is independently alkyl, alkenyl,alkynyl, alkoxy, acyl, acyloxy, carboxylic acid, ester, amine, amide,carbonate, carbamate, nitro, thioether, thioester, cycloalkyl,heteroalkyl, heterocyclyl, monosaccharide, aryl, or heteroaryl, any ofwhich is substituted or unsubstituted, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, or H;

R⁴, R⁷ and R⁸ are alkoxy, hydroxyl or H;

W¹ is O or S; or

a salt thereof.

In some aspects, X¹ can be O or S; each of R¹, R², R³, R⁹, and R¹⁰ canbe independently alkoxy, cycloalkyl, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, or H; and each of R⁴, R⁷ and R⁸ can bealkoxy, hydroxyl or H.

In some aspects, X¹ can be O; each of R¹, R², R³, R⁹, and R¹⁰ can beindependently alkoxy, cycloalkyl, halogen, hydroxyl, sulfhydryl, nitro,nitroso, cyano, azido, or H; and each of R⁴, R⁷ and R⁸ can be alkoxy,hydroxyl or H.

In yet other aspects, X¹ can be O; each of R¹ and R² can beindependently hydroxyl or H; each of R³, R⁹ and R¹⁰ can be cycloalkyl,heterocyclyl, hydroxyl, or H; R⁴ can be hydroxyl; and R⁷ and R⁸ can behydroxyl or H.

In yet other aspects, X¹ can be O; R¹ can be hydroxyl; each of R² and R³can be independently hydroxyl or H; R⁹ and R¹⁰ can be H; R⁴ can behydroxyl; and R⁷ and R⁸ can be hydroxyl or H.

In yet other aspects, X¹ is O; R¹ is hydroxyl; each of R² and R³ can beindependently hydroxyl or H; R⁹ can be heterocyclyl or H; of R¹⁰ is H;R⁴ can be independently hydroxyl or H; and each of R⁷ and R⁸ can beindependently hydroxyl or H.

In yet other aspects, X¹ is O; R¹ is hydroxyl; each of R² and R⁹ can beindependently hydroxyl or H; R³ can be cycloalkyl, hydroxyl or H; R¹⁰ isH; R4 is hydroxyl; and each of R and R⁸ can be independently hydroxyl orH. In one embodiment, cycloalkyl of R³ can be a monosaccharide.

In a particular example, the compound can be of the following formula:

In a particular example, the compound can be myricetin. In oneparticular example, the compound can be robinetin. In one particularexample, the compound can be tricetin. In one particular example, thecompound can be 7,3′,4′,5′-tetrahydroxyflavone. In one particularexample, the compound can be ficetin. In one particular example, thecompound can be kaempferol. In one particular example, the compound canbe quercetin.

In a particular example, a protective agent within the pharmaceuticalcomposition can be a compound with the following structure:

In a particular example, a protective agent within the pharmaceuticalcomposition can be vitexin, wherein vitexin has the following structure:

In a particular example, the compound may be a compound according toFormula 1, wherein R¹ is hydroxyl, R² is hydroxyl, R³ is monosaccharide,R⁴ is hydroxyl, R⁷ is hydroxyl, R⁸ is hydroxyl, R⁹ is H, R¹⁰ is H, X¹ isO, and W¹ is O, or a salt thereof. In a particular example, the compoundcan be of the following formula:

In a particular example, the compound can be myricetrin/myricitrin.

In some cases, the monosaccharide can be a natural or unnatural sugarmolecule. Non-limiting examples of a monosaccharide include glucose,dextrose, fructose, galactose mannose, ribose, deoxyribose, D-allose,L-allose, D-altrose, L-altrose, D-fucose, L-fucose, D-gulose, L-gulose,D-sorbose, D-tagatose, D-arabinose, L-arabinose, D-lyxose, L-lyxose,rhamnose, D-ribose, ribulose, sucroribulose, D-xylose, D-erythrose,L-erythrose, erythrulose, D-threose, and L-threose.

In some cases, the pharmaceutical compositions described herein maycomprise a compound according to Formula 2,

wherein:X¹ is CR⁵R⁶, NR⁵, O, S, C═O, or C═S;

represents a single or double bond;each of R¹, R², R³, R⁵, R⁶, R⁹, and R¹⁰ is independently alkyl, alkenyl,alkynyl, alkoxy, acyl, acyloxy, carboxylic acid, ester, amine, amide,carbonate, carbamate, nitro, thioether, thioester, cycloalkyl,heteroalkyl, heterocyclyl, monosaccharide, aryl, or heteroaryl, any ofwhich is substituted or unsubstituted, halogen, hydroxyl, sulfhydryl,nitro, nitroso, cyano, azido, or H;R⁴, R⁷ and R⁸ are hydroxyl;W¹ is O or S;or a salt thereof.

In one particular example, the pharmaceutical compositions Formula 2 maycomprise a dihydrorobinetin.

In some cases, the cancer treatment within the pharmaceuticalcomposition described herein may be a chemotherapeutic drug (e.g.,anthracyclines, protein kinase inhibitors, and proteasome inhibitors).Generally, the chemotherapeutic drug may be a drug that can inducecardiotoxicity in a patient or subject. Non-limiting examples of ananthracycline may include daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, or valrubicin. Non-limiting examples of aprotein kinase inhibitor may include a tyrosine kinase inhibitor,afatinib, axitinib, bosutinib, cabozantinib, carfilzomib, ceritinib,cobimetanib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus,gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,nintedanib, osimertinib, palbociclib, pazopanib, pegaptanib, ponatinib,regorafenib, ruxolitinib, sirolimus, sorafenib, sunitinib, tofacitinib,tofacitinib, temsirolimus, trametinib, vandetanib, vemurafenib, orvismodegib. Non-limiting examples of tyrosine kinase inhibitors thatcause cardiotoxicity include dasatinib, imatinib, lapatinib, mesylate,nilotinib, sorafenib and sunitinib. Non-limiting example of proteasomeinhibitors include bortezomib.

In some cases, a pharmaceutical composition disclosed herein maycomprise a co-formulation of an anthracycline (e.g., doxorubicin) and acompound of Formula 1 or Formula 2 (e.g., myricetin, vitexin, robinetin,tricetin, ficetin, 7,3′,4′,5′-tetrahydroxyflavone, dihydrorobinetin,myricitrin, and/or a derivative or salt thereof). For example, thepharmaceutical composition comprises a co-formulation of doxorubicin andmyricetin. In another example, the pharmaceutical composition disclosedherein may comprise a co-formulation of a protein kinase inhibitor orproteasome inhibitor (e.g., afatinib or bortezomib) and myricetin. Inanother example, the pharmaceutical composition disclosed herein maycomprise a co-formulation of tyrosine kinase inhibitor and a protectiveagent. In one embodiment, the pharmaceutical composition disclosedherein may comprise a co-formulation of sunitinib and myricetin. Inanother example, the pharmaceutical composition disclosed herein maycomprise a co-formulation of sorafenib and myricetin.

In some cases, the cancer treatment within the pharmaceuticalcomposition described herein may be a biologic agent (e.g., anantibody). Non-limiting examples of a biologic agent includeadotrastuzumabemtansine, alemtuzumab, bevacizumab, blinatumomab,brentuximab vedotin, catumaxomab, cetuximab, gemtuzumab ozogamicin,ibritumomab tiuxetan, ipilimumab, necitumumab, nivolumab, obinutuzumab,ofatumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab,rituximab, tositumomab-I131, or trastuzumab. For example, apharmaceutical composition disclosed herein may comprise aco-formulation of bevacizumab and myricetin. For example, apharmaceutical composition disclosed herein may comprise aco-formulation of trastuzumab and myricetin.

The compounds of the current disclosure, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat are defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids. The present invention is meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. A “stereoisomer” refers to a compound made up ofthe same atoms bonded by the same bonds but having differentthree-dimensional structures, which are not interchangeable. The presentdisclosure contemplates various stereoisomers and mixtures thereof andincludes “enantiomers”, which refers to two stereoisomers whosemolecules are nonsuperimposeable mirror images of one another. Opticallyactive (+) and (−), (R)- and (S)-, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, for example, chromatography and fractionalcrystallization. Conventional techniques for the preparation/isolationof individual enantiomers include chiral synthesis from a suitableoptically pure precursor or resolution of the racemate (or the racemateof a salt or derivative) using, for example, chiral high pressure liquidchromatography (HPLC). When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers.

When desired, the (R)- and (S)-isomers of the compounds of the presentdisclosure, if present, may be resolved by methods known to thoseskilled in the art, for example by formation of diastereoisomeric saltsor complexes which may be separated, for example, by crystallization;via formation of diasteroisomeric derivatives which may be separated,for example, by crystallization, gas-liquid or liquid chromatography;selective reaction of one enantiomer with an enantiomer-specificreagent, for example enzymatic oxidation or reduction, followed byseparation of the modified and unmodified enantiomers; or gas-liquid orliquid chromatography in a chiral environment, for example on a chiralsupport, such as silica with a bound chiral ligand or in the presence ofa chiral solvent. Alternatively, a specific enantiomer may besynthesized by asymmetric synthesis using optically active reagents,substrates, catalysts or solvents, or by converting one enantiomer tothe other by asymmetric transformation.

Compounds may be dosed in their enantiomerically pure form. In someexamples, the compound has an enantiomeric excess greater than about50%, 60%, 70%, 80%, 90%/0, 95%, 96%, 97%, 98%, or 99%, Compounds may bedosed in their diasteriomerically pure form. In some examples, thecompound has a diasteriomeric excess greater than about 500%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, or 99%.

Stereocenters may be defined using the Cahn-Ingold-Prelog priorityrules. Compounds may have stereocenters in the R-configuration.Compounds may have stereocenters in the S-configuration.

Some compounds may exhibit polymorphism. It is to be understood that thepresent disclosure encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, of a compoundof the disclosure, which possesses the useful properties describedherein, it being well known in the art how to prepare optically activeforms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

In certain particular embodiments, more than one compound of the currentdisclosure may be administered at a time to a subject. In someembodiments, two compounds of the current disclosure in combination mayact synergistically or additively, and either compound may be used in alesser amount than if administered alone.

In certain embodiments, compounds disclosed herein and/or pharmaceuticalcompositions thereof can be used in combination therapy with othertherapeutic agents. The compounds disclosed herein and/or pharmaceuticalcompositions thereof and the therapeutic agent can act additively or,more preferably, synergistically. In some embodiments, compoundsdisclosed herein and/or pharmaceutical compositions thereof areadministered concurrently with the administration of another therapeuticagent. For example, compounds disclosed herein and/or pharmaceuticalcompositions thereof may be administered together with anothertherapeutic agent. In other embodiments, compounds disclosed hereinand/or pharmaceutical compositions thereof are administered prior orsubsequent to administration of other therapeutic agents.

The compounds of the present disclosure, or their pharmaceuticallyacceptable salts, are generally administered in a therapeuticallyeffective amount. The amount of the compound actually administered maybe determined by a physician or caregiver, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the compound administered and its relative activity,the age, weight, the response of the individual patient, the severity ofthe patient's symptoms, and the like.

The present disclosure further provides salts of any compound describedherein. The term “salt” refers to salts derived from a variety oforganic and inorganic counter ions well known in the art. Salts include,for example, acid-addition salts and base-addition salts. The acid thatis added to a compound to form an acid-addition salt can be an organicacid or an inorganic acid. Inorganic acids from which salts can bederived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. A base that is added to a compound to form a base-addition saltcan be an organic base or an inorganic base. In some cases, a salt canbe a metal salt. In some cases, a salt can be an ammonium salt.Inorganic bases from which salts can be derived include, for example,sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum, and the like. Organic bases from whichsalts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike.

Acid addition salts can arise from the addition of an acid to a compounddescribed herein. In some cases, the acid can be organic. In some cases,the acid can be inorganic. Non-limiting examples of suitable acidsinclude hydrochloric acid, hydrobromic acid, hydroiodic acid, nitricacid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid,nicotinic acid, isonicotinic acid, lactic acid, salicylic acid,4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisinic acid,gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoicacid, glutamic acid, pantothenic acid, acetic acid, propionic acid,butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid,maleic acid, hydroxymaleic acid, methylmaleic acid, glycolic acid, malicacid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, phenylacetic acid,N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonicacid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid,2-phosphoglyceric acid, 3-phosphoglyceric acid, glucose-6-phosphoricacid, and an amino acid. Non-limiting examples of suitable acid additionsalts include a hydrochloride salt, a hydrobromide salt, a hydroiodidesalt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, aphosphate salt, a hydrogen phosphate salt, a dihydrogen phosphate salt,a carbonate salt, a bicarbonate salt, a nicotinate salt, anisonicotinate salt, a lactate salt, a salicylate salt, a4-aminosalicylate salt, a tartrate salt, an ascorbate salt, agentisinate salt, a gluconate salt, a glucaronate salt, a saccaratesalt, a formate salt, a benzoate salt, a glutamate salt, a pantothenatesalt, an acetate salt, a propionate salt, a butyrate salt, a fumaratesalt, a succinate salt, a citrate salt, an oxalate salt, a maleate salt,a hydroxymaleate salt, a methylmaleate salt, a glycolate salt, a malatesalt, a cinnamate salt, a mandelate salt, a 2-phenoxybenzoate salt, a2-acetoxybenzoate salt, an embonate salt, a phenylacetate salt, anN-cyclohexylsulfamate salt, a methanesulfonate salt, an ethanesulfonatesalt, a benzenesulfonate salt, a p-toluenesulfonate salt, a2-hydroxyethanesulfonate salt, an ethane-1,2-disulfonate salt, a4-methylbenzenesulfonate salt, a naphthalene-2-sulfonate salt, anaphthalene-1,5-disulfonate salt, a 2-phosphoglycerate salt, a3-phosphoglycerate salt, a glucose-6-phosphate salt, and an amino acidsalt.

Metal salts can arise from the addition of an inorganic base to acompound described herein. The inorganic base consists of a metal cationpaired with a basic counterion, such as, for example, hydroxide,carbonate, bicarbonate, or phosphate. The metal can be an alkali metal,alkaline earth metal, transition metal, or main group metal.Non-limiting examples of suitable metals include lithium, sodium,potassium, caesium, cerium, magnesium, manganese, iron, calcium,strontium, cobalt, titanium, aluminium, copper, cadmium, and zinc.Non-limiting examples of suitable metal salts include a lithium salt, asodium salt, a potassium salt, a caesium salt, a cerium salt, amagnesium salt, a manganese salt, an iron salt, a calcium salt, astrontium salt, a cobalt salt, a titanium salt, an aluminium salt, acopper salt, a cadmium salt, and a zinc salt. Ammonium salts can arisefrom the addition of ammonia or an organic amine to a compound describedherein. Non-limiting examples of suitable organic amines includetriethyl amine, diisopropyl amine, ethanol amine, diethanol amine,triethanol amine, morpholine, N-methylmorpholine, piperidine,N-methylpiperidine, N-ethylpiperidine, dibenzyl amine, piperazine,pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, pipyrazine,ethylenediamine, N,N′-dibenzylethylene diamine, procaine,chloroprocaine, choline, dicyclohexyl amine, and N-methylglucamine.Non-limiting examples of suitable ammonium salts can be a triethyl aminesalt, a diisopropyl amine salt, an ethanol amine salt, a diethanol aminesalt, a triethanol amine salt, a morpholine salt, an N-methylmorpholinesalt, a piperidine salt, an N-methylpiperidine salt, anN-ethylpiperidine salt, a dibenzyl amine salt, a piperazine salt, apyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt,a pyrazine salt, a pipyrazine salt, an ethylene diamine salt, anN,N′-dibenzylethylene diamine salt, a procaine salt, a chloroprocainesalt, a choline salt, a dicyclohexyl amine salt, and a N-methylglucaminesalt.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions of thedisclosure is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

The term “pharmaceutically acceptable excipient” is intended to includevehicles and carriers capable of being co-administered with a compoundto facilitate the performance of its intended function. The use of suchmedia for pharmaceutically active substances is well known in the art.Examples of such vehicles and carriers include solutions, solvents,dispersion media, delay agents, emulsions and the like. Any otherconventional carrier suitable for use with the multi-binding compoundsalso falls within the scope of the present disclosure.

In making the compositions of this disclosure, the active ingredient canbe diluted by an excipient. Some examples of suitable excipients includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, PEG, polyvinylpyrrolidone, cellulose, water,sterile saline, syrup, and methyl cellulose. The formulations canadditionally include: lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying and suspendingagents; preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of thedisclosure can be formulated so as to provide quick, sustained ordelayed release of the active ingredient after administration to thepatient by employing procedures known in the art.

In some cases, the pharmaceutical compositions described herein maycomprise an excipient that can provide long term preservation, bulk up aformulation that contains potent active ingredients, facilitate drugabsorption, reduce viscosity, add flavoring, or enhance the solubilityof the pharmaceutical composition. Non-limiting examples of excipientscan include anti-adherents, binders (e.g., sucrose, lactose, starches,cellulose, gelatin, or polyethylene glycol), coatings (e.g.,hydroxypropyl methylcellulose or gelatin), disintegrants, dyes, flavors(e.g., mint, peach, raspberry, or vanilla), glidants, lubricants,preservatives (e.g., acids, esters, phenols, mercurial compounds, orammonium compounds), sorbents, or vehicles (e.g., petroleum or mineraloil).

Formulations

The pharmaceutical compositions disclosed herein may be any type offormulation including solid formulations comprising a compound ofFormula 1 or Formula 2.

In some cases, the solid formulation comprises at least 0.01 mg, 0.1 mg,1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mug, 200 mg,250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg,700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg of one ormore protective agent of Formula 1 or Formula 2 formulated singly or incombination with a chemotherapeutic drug or biologic.

In some cases, the solid formulation may comprise at least 0.1 mg, 1 mg,2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg,300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg,750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 g, 5 g, 10 g, 25 g, 50 g or100 g of one or more protective agents (e.g., myricetin, and/or aderivative or salt thereof). For example, a pharmaceutical compositiondescribed herein may be a 100 mg solid co-formulation of myricetin (75 gof the 100 mg dose) and doxorubicin (25 mg of the 100 mg dose).

In some cases, the solid formulation (or other type of formulation) cancomprise at least 0.1 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg,100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg,550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg,or 1000 mg of dexrazoxane. For example, a pharmaceutical compositiondescribed herein may comprise a 100 mg solid co-formulation of myricetin(75 mg of the 100 mg dose) and dexrazoxane (25 mg of the 100 mg dose).

The pharmaceutical compositions disclosed herein may be a liquidformulation. In some cases, the liquid formulation can comprise at least0.1 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200mg/ml, 250 mg/ml, 300 mg/ml, 350 mg/ml, 400 mg/ml, 450 mg/ml, 500 mg/ml,550 mg/ml, 600 mg/ml, 650 mg/ml, 700 mg/ml, 750 mg/ml, 800 mg/ml, 850mg/ml, 900 mg/ml, 950 mg/ml, or 1000 mg/ml concentration of one or moreprotective agent(s) of Formula 1 or Formula 2 formulated singly or incombination with either a chemotherapeutic drug or biologic agent. Forexample, a pharmaceutical composition described herein may comprise a100 mg/mL concentration of the protective agent myricetin and a 50 mg/mLconcentration of doxorubicin.

In some cases, the liquid formulation may comprise at least 0.1 mg/ml, 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml,300 mg/ml, 350 mg/ml, 400 mg/ml, 450 mg/ml, 500 mg/ml, 550 mg/ml, 600mg/ml, 650 mg/ml, 700 mg/ml, 750 mg/ml, 800 mg/ml, 850 mg/ml, 900 mg/ml,950 mg/ml, or 1000 mg/ml concentration of myricetin, or derivative orsalt thereof. For example, a pharmaceutical composition described hereinmay comprise 100 mg/mL concentration of myricetin.

In some cases, the liquid formulation can comprise at least 0.1 mg/ml, 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml,300 mg/ml, 350 mg/ml, 400 mg/ml, 450 mg/ml, 500 mg/ml, 550 mg/ml, 600mg/ml, 650 mg/ml, 700 mg/ml, 750 mg/ml, 800 mg/ml, 850 mg/ml, 900 mg/ml,950 mg/ml, or 1000 mg/ml concentration of dexrazoxane co-formulated withone or more protective agent.

In some cases, a pharmaceutical composition described herein maycomprise at least 2 protective agents. The molar ratio of one protectiveagent to at least one other protective agent can be about 1:1, about1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8,about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50,about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about1:1,000, about 1:10,000, or about 1:>10,000.

In some cases, a pharmaceutical composition described herein maycomprise a cancer treatment (e.g., chemotherapeutic drug or biologicagent) and at least one protective agent. The molar ratio of the cancertreatment to at least one other protective agent can be about >10,000:1,about 10,000:1, about 1,000:1, about 100:1, about 90:1, about 80:1,about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1,about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about1:80, about 1:90, about 1:100, about 1:1,000, about 1:10,000, or about1:>10,000

Kits

In some cases, the pharmaceutical compositions disclosed herein may beassembled into kits. In some cases, the kit can comprise a protectiveagent, wherein the protective agent may exist as distinct entitieswithin the kit or as a co-formulation. For example, the kit may compriseone or more protective agents selected from the group consisting ofmyricetin, tricetin, robinetin, ficetin, vitexin, dihydrorobinetin,7,3′,4′,5′-tetrahydroxyflavone, myricitrin, and dexrozoxane. In somecases, the kit can comprise at least two protective agents, wherein thetwo protective agents may exist as distinct entities within the kit oras a co-formulation. For example, the kit may comprise at least twoprotective agents selected from the group consisting of myricetin,tricetin, robinetin, ficetin, vitexin, dihydrorobinetin,7,3′,4′,5′-tetrahydroxyflavone, myricitrin, and dexrozoxane. In aparticular example, the kit may comprise a co-formulation of myricetinand dexrazoxane. In some cases, the kit can comprise a cancer treatmentand at least one protective agent, wherein the cancer treatment and atleast one protective agent may exist as distinct entities within the kitor as a co-formulation. For example, the kit may comprise a cancertreatment and myricetin and/or a derivative thereof. For example, thekit may comprise a cancer treatment and robinetin and/or a derivativethereof. For example, the kit may comprise a cancer treatment anddihydrorobinetin and/or a derivative thereof. For example, the kit maycomprise a cancer treatment and tricetin and/or a derivative thereof.For example, the kit may comprise a cancer treatment and ficetin and/ora derivative thereof. For example, the kit may comprise a cancertreatment and 7,3′,4′,5′-tetrahydroxyflavone and/or a derivativethereof.

In one embodiment, the kit may comprise a co-formulation of doxorubicinand myricetin.

In some cases, the kit may also comprise instructions for use. The kitmay also comprise vials, tubes, needles, packaging, or other materials.

Kits with unit doses of one or more of the compounds described herein,usually in oral or injectable doses, are provided. Such kits may includea container containing the unit dose, an informational package insertdescribing the use and attendant benefits of the drugs in treating thedisease, and optionally an appliance or device for delivery of thecomposition.

The kit may further comprise any device suitable for administration ofthe composition. For example, a kit comprising an injectable formulationof pharmaceutical compositions may comprise a needle suitable forsubcutaneous administration and an alcohol wipe for sterilization of theinjection site.

In some cases, kits may be provided with instructions. The instructionsmay be provided in the kit or they may be accessed electronically. Theinstructions may provide information on how to use the compositions ofthe present disclosure. The instructions may further provide informationon how to use the devices of the present disclosure. The instructionsmay provide information on how to perform the methods of the disclosure.In some cases, the instructions may provide dosing information. Theinstructions may provide drug information such as the mechanism ofaction, the formulation of the drug, adverse risks, contraindications,and the like. In some cases, the kit is purchased by a physician orhealth care provider for administration at a clinic or hospital. In somecases, the kit is purchased by a laboratory and used for screeningcandidate compounds.

EXAMPLES Example 1. Myricetin Provides Long-Term Cardioprotection (CellViability)

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at day 3, before performingexperiments. Samples were either mock-treated, treated with 1.25 μMdoxorubicin, treated with myricetin, or co-treated with 1.25 μM ofdoxorubicin and myricetin for 72 hours. Following treatment, the sampleswere incubated Hoeschst 33342 to indicate cell nuclei. Cells were imagedusing the INCell Analyzer2200, and images were analyzed to quantify thetotal number of cells and plotted as a percentage of total cellsnormalized to control (left), where each data point was obtained fromthree biological replicates. Representative images (FIG. 3 , right) arepresented for each sample, where an increase in Hoechst 33342 signalrepresents an increase ion cell viability.

Cardiomyocytes were either mock-treated, treated with 1.25 μMdoxorubicin, treated with myricetin, or co-treated with 1.25 μM ofdoxorubicin and myricetin for 72 hours, and subsequently stained todetect total number of cells (FIG. 3 ). Myricetin was a potent protectorof cell viability. Cardiomyocytes treated with 1.25 μM doxorubicin, inthe absence of myricetin, exhibited a 62.6% reduction in the number oftotal cells, whereas cardiomyocytes co-treated with myricetin and 1.25μM doxorubicin exhibited a 27.57% reduction the number of total cells,as compared to mock-treated control. Cardiomyocytes treated withmyricetin, in the absence of doxorubicin, exhibited no significantdifference in the number of total cells, as compared to mock-treatedcontrol. Error bars represent standard deviation. Representative imagesare presented for each sample, where an increase in Hoechst 33342 signalrepresents an increase in cell viability. Cardiomyocytes treated with1.25 μM doxorubicin (FIG. 3 , right: bottom left panel), in the absenceof myricetin, exhibited a reduction in Hoechst 33342 signal, whereascardiomyocytes co-treated with 79 μM myricetin and 1.25 μM doxorubicin(FIG. 3 , right: bottom right panel) exhibited less reduction in Hoechst33342 signal, as compared to mock-treated control (FIG. 3 , right: topleft panel). Cardiomyocytes treated with myricetin (FIG. 3 , right: topright panel), in the absence of doxorubicin, exhibited no significantdifference in Hoechst 33342 signal, as compared to mock-treated control.

Example 2. Effects of Myricetin on Doxorubicin-Induced Cardiotoxicity 2Days Following Treatment (Mitochondrial Toxicity)

Human iPSC-derived cardiomyocytes were prepared by differentiatinginduced pluripotent stem cells into cardiomyocytes. Cells were culturedfor 4 days post-differentiation, changing media at day 3, beforeperforming experiments. Cardiomyocytes were treated with 1.25 μMdoxorubicin (FIG. 4A), or co-treated with 1.25 μM of doxorubicin and 79μM myricetin (FIG. 4B) for 2 days. Following treatment, the samples wereincubated with a tetramethylrhodamine methyl ester (TMRM) dye toindicate mitochondrial health, and Hoechst 33342 to identify cellnuclei. Cells were imaged using the INCell Analyzer2200. Representativeimages are presented for each sample, wherein a decrease in TMRM signalindicates an increase in mitochondrial toxicity. Myricetin was a potentprotector against doxorubicin-induced mitochondrial toxicity, asindicated by a greater TMRM signal in cells co-treated with 1.25 μMdoxorubicin and 79 μM myricetin (FIG. 4B) as compared to cells treatedwith 1.25 μM doxorubicin in the absence of myricetin (FIG. 4A).

Example 3. Effects of Myricetin on Doxorubicin-Induced Cardiotoxicity 3Days Following Treatment (Contractility)

Human iPSC-derived cardiomyocytes were prepared as described above.Samples were either mock-treated, treated with 1.25 μM doxorubicin,treated with 79 μM myricetin, or co-treated with 1.25 μM of doxorubicinand 79 μM myricetin for 72 hours. Following treatment, videos of beatingcardiomyocytes were captured using Pulse, and analyzed to quantify beatrate (FIG. 5 ; left) from plots of cell contraction, where each datapoint was obtained from three biological replicates. Representativeplots of cell contraction (FIG. 5 ; right) are presented for eachsample. Myricetin was a potent protector of cell contractility.Mock-treated cardiomyocytes contracted at 33.33 beats per minute,whereas treatment with 1.25 μM doxorubicin, in the absence of myricetin,completely inhibited contraction. Cardiomyocytes treated with myricetin,or co-treated with myricetin and 1.25 μM doxorubicin, contracted at39.33 or 37.33 beats per minute, respectively. FIG. 6A-C depicts a chartproviding the raw data (6A) or normalized data (6B) for the experimentsdepicted in FIG. 3 , or the raw data for the experiments depicted inFIG. 5 (6C).

Example 4. Effects of Various Flavonols and Flavones onDoxorubicin-Induced Cardiotoxicity 3 Days Following Treatment(Apoptosis)

Cardiomyocytes were prepared as described above. Cells were co-treatedwith 1 μM of doxorubicin and either myricetin (FIG. 7A), myricetrin(FIG. 7B), or dihydromyricetin (FIG. 7C) for 3 days. Followingtreatment, the samples were incubated with a CellEvent dye to indicateapoptosis-positive cells, and a second dye to identify cell nuclei.Cells were imaged using the INCell Analyzer2200, and images wereanalyzed to quantify the percentage of apoptotic cells. Data arepresented from two independent sets of screening where each data pointwas obtained from triplicate.

Cardiomyocytes co-treated with doxorubicin and either myricetin (FIG.7A), myricitrin (FIG. 7B), or dihydromyricetin (FIG. 7C) exhibitedprotective effects against apoptosis, with half minimal inhibitoryconcentrations (IC50; e.g., the drug concentration that induces 50percent apoptosis) of 20.46 μM, 38.48 μM, 40.48 μM, respectively.

Example 5. Myricetin Reduces DOX's Cytotoxicity in Cardiomyocytes

To assess the effect of MYR against DOX-induced cytotoxicity, humaniPSC-derived cardiomyocytes were mock-treated (triangle) or treated with100 μM of myricetin (MYR; circle) and increasing concentrations ofdoxorubicin (DOX) for 72 hours, and then incubated with dyes thatindicate mitochondrial health (TMRM, Life Technologies) and cellularnuclei (Hoechst33342, Life Technologies). Cells were imaged using INCellAnalyzer2200 (GE). Total number of healthy cells were counted andplotted as percentage of mock-treatment control. Lethal concentration atwhich 50% of cells were killed (LC50) by doxorubicin was shifted from0.41 μM in mock-treated to 1.29 μM in MYR-treated conditions for iPSCcardiomyocytes (FIG. 8 ). Data are presented from multiple independentsets of screening where each data point was obtained from triplicate.(n=3). Y-axis: percentage of cell survival; and X-axis: increasingconcentrations of DOX (FIG. 8 ).

Example 6. Myricetin Protects Against DOX-Induced Cell Death inCardiomyocytes

To measure the rescue rates from the DOX-induced cell death incardiomyocytes, the protective effect of myricetin was directly comparedwith that of dexraxozane (DEX; standard of care). Human iPSC-derivedcardiomyocytes were treated with 0.5 μM of Doxorubicin and increasingconcentrations of myricetin (MYR, circle) or dexraxozane (DEX, square).After 72 hours of treatment, cells were incubated with dyes thatindicate mitochondrial health (TMRM, Life Technologies) and cellularnuclei (Hoechst33342, Life Technologies). Cells were imaged using INCellAnalyzer2200 (GE). Total number of healthy cells were counted andplotted as percentage of doxorubicin-treatment control. Half maximaleffective concentration (EC50) for MYR was 7.50 μM (FIG. 9 ). Incontrast, DEX did not exhibit any significant rescues from DOX-inducedcytotoxicity. (n=3).

Example 7. Myricetin Protects Against DOX-Induced ContractilityDysfunction and DNA Double Strand Break in Cardiomyocytes

To assess the protective effect of myricetin on the contractility ofheart cells, cell samples were prepared by differentiating inducedpluripotent stem cells into cardiomyocytes. Cells were cultured for 4days post-differentiation, changing media at day 3, before performingexperiments. Human iPSC-derived cardiomyocytes were then treated withDMSO, DOX (0.5 μM), DOX plus DEX (100 μM), or DOX plus MYR (100 μM).After 48 hours of treatment, videos of beating cardiomyocytes werecaptured with Pulse (Cellogy). DOX treatment induced dysfunction incardiomyocyte contraction as evidenced by reduction in beating,duration, and peak height. This contractile dysfunction wassignificantly corrected by MYR as compared to DEX (FIG. 10 ). Data arepresented from multiple independent sets of experiments where each datapoint was obtained from 6 samples (n=6). Student T-Test (unpaired,two-tailed) was used to determine the significance of the difference.

To determine whether MYR protects against DOX-induced DNA double strandbreak in cells, human iPSC-derived cardiomyocytes were treated withDMSO, DOX (0.5 μM), DOX plus DEX (100 μM), or DOX plus MYR (100 μM).After 48 hours of treatment, cells were immunostained with antibodyagainst γH2AX (EMD Millipore) to detect double strand break. Cells werethen imaged using INCell Analyzer2200 (GE) and percentages ofγH2AX-positive cells were quantified for each condition. While DEXexhibited little or no protection against DOX-induced double strandbreak in the tested heart cells, MYR conferred significant protectionfrom DOX-related DNA damage (FIG. 11 ). Student T-Test (unpaired,two-tailed; n=6).

Example 8. MYR Protects Against Sarcomere Disruption by DOX

DOX-induced cell death is often manifested by severity of structuraldisruptions of cardiomyocyte organization (e.g., sarcomere). To assessthe protective effect of MYR against DOX-induced sarcomere disruption,human iPSC-derived cardiomyocytes were treated with DMSO, DOX (0.5 μM),or DOX plus MYR (100 μM). After 72 hours of treatment, cells wereimmunostained with antibody against Cardiac Troponin T (Abcam) to showsarcomeric organization in of the heart cells. As shown in FIG. 12 , MYRconferred significant protection against DOX-induced sarcomeredisruption in cardiomyocytes, suggesting that the protective effects ofMYR against DOX-induced cell death are well manifested by the structuralintegrity of the cardiomyocytes.

Example 9. Myricetin is a Potent Inhibitor of TOPOIIα and β

To gain insights into a molecular mechanism of myricetin (MYR) and thatof dexraxozane (DEX) on cardioprotection, the effect of these twocompounds on topoisomerases II (i.e., TOPOIIα and β), an apparent targetof DOX, was assessed.

200 ng of kinetoplast DNA (kDNA) was incubated with one enzymatic unitof TOPOIIα or TOPOIIβ enzyme (Inspiralis) and with variousconcentrations of MYR or DEX at 37° C. for 30 min. The reaction was thenseparated on 1% agarose gel for visualization of decatenated DNA (bottomband). The efficiency of catalytic inhibition was quantified bymeasuring the relative intensity of the band.

MYR and DEX exhibited 50% inhibition (IC50) of TOPOIIα enzyme activityat concentrations of 1.18 μM and 52.7 μM, respectively (FIG. 13 ; n=3).IC50 of TOPOIIβ enzyme activity for MYR and DEX were 2.07 μM and 34.43μM, respectively (FIG. 13 ; n=3). The data suggest that MYR is asignificantly more potent inhibitor than DEX for both topoisomerases IIαand β.

Example 10. Unlike DEX, MYR does not Induce TOPOII Protein Degradation

To further distinguish molecular mechanisms of MYR from those of DEX andalso to determine whether the inhibitory effects of MYR on TOPOIIobserved in the decatenation assays above is due to degradation ofTOPOII proteins, human iPSC-derived cardiomyocytes were treated withDMSO, DEX (100 μM), or MYR (100 μM) for 24 hours, and immunostained withantibody against topoisomerase IIβ (BD Biosciences).

Cells were imaged using INCell Analyzer2200 (GE) and topoisomerase 111protein levels were quantified. Student T-Test (unpaired, two-tailed)was used to determine the significance of the difference.

As shown in FIG. 14 , treatment with DEX resulted in markeddisappearance of TOPOIIβ in iPSC-CMs, whereas MYR exerted no effect ontopoisomerase IIβ protein levels (FIG. 14 ) (n=3). The results confirmedthe hypothesis that DEX can negatively affect the stability oftopoisomerases IIβ (TOPOIIβ), which may lead to the depletion of theseenzymes from the heart cells, effectively resulting in prevention of DNAdamage generated by poisonous effects on these enzymes by theanthracycline. These results also confirmed that the mechanism by whichMYR confer protection from anthracycline-induced toxicity is entirelyindependent and can be distinguished from that of DEX. Further, theeffect of MYR observed in topoisomerase inhibition is not due to TOPOIIβprotein degradation or depletion of the enzyme from DOX's debilitatingeffects on the heart cells. It can be concluded that inhibition oftopoisomerase II activity, particularly without affecting the stabilityof TOPOII enzymes, is an important factor for MYR's ability to confercardioprotection.

Example 11. Neither DHM Nor DHR Inhibit TOPOIIα or TOPOIIβ

Since the ability of MYR to confer cardioprotection against DOX-inducedtoxicity is independent from DEX, it was further investigated todetermine whether other flavonoid compounds have a similar effect ontopoisomerase II activity like MYR.

First, MYR (flavonol) and dihydromyricetin (flavanonol) were tested fortheir inhibitory effect on topoisomerase II enzymatic function.Dihydromyricetin (DHM) shares a similar chemical structure except forthe presence of a single bond in the major C-ring of the flavonoidscaffold.

200 ng of Kinetoplast DNA (kDNA) was incubated with one enzymatic unitof TOPOIIβ and different concentrations of MYR (circle) or DHM(triangle) at 37° C. for 30 min (FIG. 15 ). The reaction was thenseparated on 1% agarose gel for visualization of decatenated DNA (bottomband) and the catalytic inhibition efficiency was quantified bymeasuring the relative intensity of the band. Surprisingly, DHM did notinhibit TOPOIIβ (n=3) (FIG. 15 ) or TOPOIIα enzymatic activity, even atextreme concentrations (>200 μM).

Further, this result on DHM was confirmed in separate experiments withdihydrorobinetin (DHR) and robinetin (ROB) in which DHR, like DHM,showed no inhibitory activity toward these topoisomerases, whilerobinetin, like MYR, displayed a high level of inhibition on bothTOPOIIβ and TOPOIIα. These data indicate that the structural differencein the C-ring of the flavone/flavonoid scaffold plays an important rolein TOPOII inhibition.

Example 12. MYR is 2-Fold More Potent in Protecting DOX-Induced CellDeath than DHM

Next, the ability of MYR to confer cardioprotection was directlycompared with that of DHM as these two compounds display distinctiveproperty in their structures and TOPOI inhibition activity. Cell sampleswere prepared by differentiating induced pluripotent stem cells intocardiomyocytes. Cells were cultured for 4 days post-differentiation,changing media at day 3, before performing experiments. HumaniPSC-derived cardiomyocytes were treated with 0.5 μM of Doxorubicin andincreasing concentrations of myricetin (MYR, circle) or dihydromyricetin(DHM, triangle). After 72 hours of treatment, cells were incubated withdyes that indicate mitochondrial health and cellular nuclei. Cells werethen imaged and total number of healthy cells were counted and plottedas percentage of Doxorubicin-treatment control as described above.

As illustrated in FIG. 16 , MYR exhibited 2-fold greater potency inprotecting DOX-induced cell death than DHM as half maximal effectiveconcentrations (EC50) for MYR and DHM were 7.50 μM and 13.96 μM,respectively. (n=3) Based on these results, it was concluded that adouble bond in C ring of the flavone/flavonoid scaffold enhances potencyfor the cardioprotective properties by conferring the inhibitory effectson topoisomerases H.

These observations were followed up by a DOX-induced DNA double strandbreak assay. Human cardiomyocytes were treated with 0.5 μM ofdoxorubicin and with increasing concentrations of MYR (circle) or DHM(triangle). After 48 hours of treatment, cells were immunostained withantibody against γH2AX (EMD Millipore) to detect DNA double strandbreak. Cells were imaged using INCell Analyzer2200 (GE) and percentagesof γH2AX-positive cells was quantified for each condition. Consistentwith its cell death rescue rate, MYR was 2-fold more potent inprotecting DOX-induced double strand break than DHM. Concentrations atwhich DOX-induced double strand break was reduced to 50% (IC50) for MYRand DHM were 5.28 μM and 11.30 μM, respectively (FIG. 17 ). (n=3)

To investigate the effect of myricetin on cardiomyocytes exposed todoxorubicin, mRNA expression levels were determined in the cells treatedwith DOX alone, myricetin alone, and DOX plus myricetin. Surprisinglywhile myricetin did not have any effect on TOPOIIβ mRNA expression byitself, DOX significantly repressed TOPOIIβ expression at 24 and 48hours (FIG. 18 ). However, in the presence of myricetin, TOPOIIexpression was restored to a level close to normal by myricetin,effectively preventing any transcription alteration by DOX (FIG. 18 ).This data suggested that there appeared to be a synergistic effectbetween DOX and myricetin on TOPOIIβ expression. With respect toexpression of TOPOIIα, DOX slowly repressed expression of TOPOIIα overtime. In the presence of DOX, however, myricetin further repressedTOPOIIα, suggesting a differential effect of myricetin on thesetopoisomerases II at molecular and cellular levels. Combined downregulatory effect of myricetin and DOX on TOPOIIα is larger than whatwas observed with DOX alone.

Example 13. Cardioprotective Properties of MYR Analogs

To further investigate the relationship between the structure (e.g.,flavone/flavonol scaffold) and biological activity (e.g.,cardioprotection, TOPOII inhibition, etc.), a group of additionalflavonoid compounds related to myricetin were identified and tested fortheir activity.

I. Identification of Flavonoids with Cardio-Protective PropertiesMediated Through TOPOII Inhibition

Anthracycline-induced cardiotoxicity occurs when the drug such asdoxorubicin intercalates the DNA upon a cleavage of DNA by topoisomeraseII enzymes, thereby effectively preventing TOPOIIα or β from ligatingthe cleaved DNA strands back together. Therefore, a working hypothesiswas proffered based on cardioprotective properties of flavonoids beingmediated through topoisomerases IIα and β (TOPOIIα and TOPOIIβ)inhibition.

A systematic study on the hydroxyl substituents of the MYR scaffold wasconducted for biological activity. The objective was to explore chemicalspace around MYR to identify which substituents (e.g., hydroxyl,alkoxyl, or heterocyclic) are required at various positions and todetermine which chemical structure(s) is the essential component forbeing a cardioprotectant.

With respect to biological activity, a biochemical decatenation assaywas used as described above to assess TOPOIIα and TOPOIIβ inhibition.Doxorubicin treated human iPSC-derived cardiomyocytes were employed tomeasure the protective effect of these analogs on cardiomyocytes.

Starting from the bare flavone, 48 myricetin analog compounds withhydroxyl substituents present or missing at the 3, 5, 7, 3′, 4′, or 5′positions were identified (myricetin is the compound with all sixhydroxyl substituents present). In addition to the 48 myricetin analogswas chromone which is devoid of the B-ring of flavone, anddihydromyricetin and dihydrorobinetin (DHR) which both lack the doublebond in the C-ring.

Because substituents can be incorporated into the flavone scaffold atpositions 8 and/or 6 on the A-ring similar to vitexin, and also atpositions 2′ and/or 6′ on the C-ring, hydroxyl, alkoxy, alkyl andheterocyclic, halides were contemplated for analysis. The study alsoincluded chemical moieties other than hydroxyl substituents present inthe MYR scaffold (Formula 1), such as alkoxy (particularly methoxy),alkyl (methyl), heterocyclic, or halides at 3, 5, 7, 3′, 4′, and/or 5′positions.

This study led to the identification of the minimum structure based onthe MYR scaffold required for end point activity. Among the compounds ofspecific combination of hydroxyl groups in 3, 5, 7, 3′, 4′, 5′ positionsselected for biological activity for cardioprotection (e.g., TOPOIIβinhibition, and DNA double strand break), a certain group of compoundswith specific combinations of substituents present or missing at the 3,5, 7, 3′, 4′, 5′ positions was found to be critical for biologicalproperties as a cardioprotectant with decreased cytotoxicity.

TABLE 1 iPSC-CM protection Max Effect EC50 TOPIIβ TOPIIα ID CompoundName (%) (μM) Toxicity Rescue Inhibition Inhibition 1 3,5,7,3′,4′,5′-hexahydroxyflavone 78 14.48 − ++++ +++ +++ (myricetin) 2 3,7,3′,4′,5′-pentahydroxyflavone 64 12.62 − ++++ +++ +++ (robinetin) 3 5,7,3′,4′,5′-pentahydroxyflavone 56 17.19 * +++ +++ +++ (tricetin) 4 3,5,7,3′,4′-pentahydroxyflavone 58 20.5 * ++ +++ +++ (quercetin) 5 3,7,3′,4′-tetrahydroxyflavone 36 16.32 * ++ +++ +++ (fisetin) 6 7,3′4′,5′- 7117.13 − +++ − − tetrahydroxyflavone 7 3,5,7,4′- tetrahydroxyflavone 4626.01 − ++ − − (kaempferol) 8 3′,4′,5′- 64 43.01 − + − −trihydroxyflavone 9 5,7,3′,4′- tetrahydroxyflavone 62 9.67 * +++ − −(luteolin) 10 3,7,4′- trihydroxyflavone 27 3.26 * + − − (resokaempferol)11 7,3′,4′- 24 6.25 * + − − trihydroxyflavone 12 3,3′,4′- 16 6.43 * + −− trihydroxyflavone 13 5,7,4′,- † − − − N/A N/A trihydroxyflavone(apigenin) 14 3′,4′- † − * − N/A N/A dihydroxyflavone 15 7,4′- † − * −N/A N/A dihydroxyflavone 16 3,4′- † − * − N/A N/A dihydroxyflavone 174′-hrydroxyflavone † − − − N/A N/A 18 3,7,3′- † − * − N/A N/Atrihydroxyflavone 19 3,5,7- † − * − N/A N/A trihydroxyflavone 20 3,7- †− * − N/A N/A dihydroxyflavone 21 7,3′- † − * − N/A N/A dihydroxyflavone22 3,3′- † − * − N/A N/A dihydroxyflavone 23 5,7- † − * − N/A N/Adihydroxyflavone 24 7-hydroxyflavone † − * − N/A N/A 25 3-hydroxyflavone† − * − N/A N/A 26 3′,5′- † − * − N/A N/A dihydroxyflavone 273′-hydroxyflavone † − * − N/A N/A 28 flavone † − * − N/A N/A 29 chromone† − − − N/A N/A 30 dihydrorobinetin 53 14.02 − +++ − − 31 3′-O-methylmyricetin 76 58.7 − + − − 32 4′-O- 68 48.6 − + − − methylmyricetin33 3′,5′-O- † − * − − − dimethylmyricetin 34 3′,4′,5′-O- † − * − − −trimethylmyricetin 35 3',4',5'-O- † − * − − − trimethylrobinetin 367,3′,4′,5′-O- † − * − − − tetramethylrobinetin 37 3,7,3′,4′,5′-O- † − *− − − pentamethylrobinetin 38 7-hydroxy-4- † − * − − − chromone+Compounds exhibited positive effects on respective biologicalproperties −Compounds exhibited negative effects on respectivebiological properties †Compounds failed to exhibit >30% protection MaxEffect at 10 μM or 100 μM on initial screen. *Compounds exhibitedcytotoxicity at 100 μM N/A, Experiment not performed as compoundsexhibited cytotoxicity and no cardioprotection activityMinimum Requirements of Hydroxyl Substituents for TOPOIIβ Inhibition andCardioprotective Effects

As shown in Table 1 above, the common features of the TOPOIIβ inhibitors(1-5) allowed an inference that hydroxyl substituents are required atpositions 3, 7, 3′, and 4′ in order for flavonoid compounds to inhibitTOPOIIβ. The only exception is tricetin (3) which does not have the3-hydroxyl substituent; all of the other four TOPOIIβ inhibitors havehydroxyl substituents at positions 3, 7, 3′, and 4′. Furthermore, thecommon features of the cardioprotective compounds (1-12) in Table 1above, allowed an additional inference that the 4′ hydroxyl substituenton the B-ring may be an essential feature, along with two of the otherthree hydroxyl substituents at positions 3, 7, and 3′, forcardioprotective activity, with the hydroxyl at position 7 preferred;the only exception being compound 8 which does not have hydroxyls atpositions 3 and 7, yet has all three hydroxyl substituents at positions3′, 4′, and 5′ on the B-ring. Moreover, considering toxicity of thetested compounds (see Table 1), one can deduce a trend thatcardioprotective compounds (1-12) which have all three 3′, 4′, and 5′hydroxyl substituents on the B-ring do not exhibit toxic effects atconcentrations less than 100 μM, whereas those cardioprotectivecompounds which have hydroxyl substituents only at positions 3′ and 4′do indeed exhibit toxic effects at concentrations less than 100 μM.Again, the one exception to this trend was tricetin (3), which exhibitssome toxic effects at concentrations less than 100 μM despite containingall three hydroxyl substituents on the B-ring. Of the twocardioprotective compounds which only have the 4′ hydroxyl substituenton the B-ring (kaempferol 7 and resokaempferol 10), kaempferol did notshow toxic effects below 100 μM, whereas resokaempferol exhibited toxiceffects at concentrations below 100 μM. Based on this analysis, it wasconcluded that:

-   -   (1) for cardioprotection, 4′ hydroxyl substituent on B-ring is        required, along with one of the following, (a) two of the three        hydroxyl substituents at positions 3, 7, and 3′, with position 7        preferred, or (b) all three hydroxyl substituents at positions        3′, 4′, and 5′ on the B-ring;    -   (2) for cardiotoxicity, 3′, 4′, and 5′ hydroxyl substituents on        the B-ring are preferable to 3′ and 4′ hydroxyl substituents on        the B-ring, to alleviate toxic effects at concentrations below        100 μM; or, 4′ hydroxyl only on the B-ring, along with all three        3, 5, and 7 hydroxyl substituents on the A/C ring system; and    -   (3) for TOPOIIβ inhibition, all four hydroxyl substituents at        positions 3, 7, 3′, and 4′ are required. Tricetin (3) does not        follow these requirements and is an outlier.        Analysis on B-Ring

It is readily apparent from the compounds listed in Table 1 that the 4′position on the B-ring requires a hydroxyl substituent forcardioprotection. Of the twelve compounds (1-12) that passed the initialscreen, all of them have the 4′-hydroxyl substituent. Moreover, of thesixteen compounds (13-28) that did not pass the initial screen, eleven(18-28) are absent the 4′-hydroxyl substituent. The remaining five4′-hydroxyl compounds (13-17) that did not pass the initial screen haveminimal substitution, e.g. only the 4′-hydroxyl as in compound 17, oronly one other hydroxyl substituent along with the 4′-hydroxyl as incompounds 14, 15, and 16. Compound 13 only has one of the requiredhydroxyl substituents from the set of 3, 7, and 3′ described above;therefore, it also does not meet the minimum requirements forcardioprotective activity. In summary, the presence of a hydroxylsubstituent at position 4′ on the B-ring is a necessary but notsufficient condition for flavonoid compounds to be cardioprotective.This structural requirement strongly hints at the presence of ahydrogen-bond between the 4′ hydroxyl on the B-ring of the protectiveagent in complex with the biological target.

1. Chromone-Related Compounds

Both chromone (29) and 7-hydroxy-4-chromone (38), which each entirelylacks the B-ring of the flavone scaffold, showed no positive effect incardiac protection. Nor did either compound confer TOPOIIβ or ainhibition (Table 1). Furthermore, 7-hydroxy-4-chromone exhibited a highlevel of cytotoxicity at 100 μM. Comparing these two B-ring nullcompounds with the corresponding tri-substituted B-ring flavone compound(8 and 6, respectively), it was concluded that the presence of theB-ring is required for cardiac protection.

Next, the observation obtained from 7-hydroxy-4-chromone was furtherexplored in 3,5,7-trihydroxyflavone having the B-ring, but lacking allB-ring substituents, 3,5,7-trihydroxyflavone exhibited neithercardioprotection nor TOPOII inhibition and displayed generalizedcytotoxicity, indicating that one or more moieties are required in theB-ring for the cardioprotection activity.

2. Methoxy Substituents on B-Ring

Since the B-ring appeared to be an essential component for biologicalactivity, compounds having the B-ring with various positionalcombinations with either hydroxyl and/or methoxy group were tested fortheir activity.

3′-O-methylmyricetin having methoxy at the 3′ position failed to inhibitTOPOII enzymes, but conferred cardioprotection without showinggeneralized cytotoxicity. However, it exhibited a significant loss ofpotency for cardioprotection (EC50, ˜59 μM). Similarly,4′-O-methylmyricetin having methoxy at 4′ position conferredcardioprotection without TOPOII inhibition. This compound displayed aloss of potency for cardioprotection (EC50, ˜48.7 μM) as compared tothat of MYR. This suggests that the presence of a single methoxysubstituent at 3′ or 4′ of the B-ring, is an important factor forcardioprotection. Confirming this observation,3′,5′-O-dimethylmyricetin, lacking a methoxy substituent at position 4but having a methoxy at positions 3′ and 5′ of the B-ring, displayedneither cardioprotection nor TOPOIIα and TOPOIIβ inhibition. Thiscompound also exhibited significant cytotoxicity. Other compounds havingmultiple methoxy replacements at positions 3′, 4′, and 5′ were alsotested for cardioprotection and TOPOII inhibition. For example, all of3′,4′,5′-O-trimethylmyricetin, 3′,4′,5′-O-trimethylrobinetin,3,7,3′,4′,5′-O-pentamethylrobinetin, 7,3′,4′,5′-O-tetramethylrobinetinentirely failed to display cardioprotection or TOPOII inhibition. Allexhibited increased levels of cytotoxicity at 100 μM.

Accordingly, replacing 4′ or 3′ hydroxyl with methoxy significantlyreduces potency and results in complete loss in TOPOII inhibition.Further, because methoxy substitution slightly enlarges and extends thecompound from the B-ring, it was postulated that having a largersubstituent extending from the B-ring, even at a marginal level, maypose a steric hindrance for the interaction between TOPOII enzyme andthe compound. Thus, hydroxyls groups in the B-ring (3′,4′,5′) appears tobe critical components that lead to cardioprotection and may play animportant role in TOPOII enzyme inhibition.

3. Quercetin and Kaempferol

Quercetin conferred cardioprotection and exhibited TOPOII inhibition.However, a high level of general cytoxicity to cardiomyocytes wasobserved at a concentration of 100 μM.

Kaempferol displayed a moderate level of cardioprotection without somelevel of cytotoxicity at 100 μM, but did not exhibit any inhibitoryeffect on TOPOIIα or TOPOIIβ. Kaempferol, however, displayed decreasedpotency and failed to achieve the maximum 50% rescue rate.

It was inferred from the data that removing 3′ (or 5′) hydroxyl may notnecessarily result in loss of TOPOII inhibition, but leads to increasedcytotoxicity and reduced potency as observed in quercetin. However,these data led to the conclusion that removing 3′ 4′, or 5′ hydroxylgroup from the B-ring result in a marked reduction in potency and/orloss of TOPOII inhibition, particularly at position 4′.

In sum, replacing one or two 3′, 4′, or 5′ hydroxyls with an alkoxy(e.g., methoxy) group renders the compound cytotoxic. Removal of 3′ and5′ hydroxyl groups from the MYR scaffold, as observed in kaempferol orremoval of either 3′ or 5′ hydroxyl as in quercetin may reduce potencyfor cardioprotection and render the compound cytotoxic. However,removing all hydroxyls on the B-ring results in complete loss ofcardioprotection and TOPOII inhibition, and causes severe cytotoxicityas observed in 3,5,7-trihydroxyflavone. Further, 4′ hydroxyl of theB-ring appears to be required for the enhanced physical attributesleading to increased potency for cardioprotection with TOPOII inhibitionand minimal cytotoxicity.

Accordingly, the preferred substituents for the B-ring are —OH in all3′, 4′ and 5′ positions in order to ensure potency and minimal toxicityas demonstrated by myricetin and robinetin.

A and C-Ring Analysis

Substituents on the heterobicyclic A/C ring system of theflavone-flavonol scaffold was assessed for the cardioprotectiveactivity. Based on the observations made on the B-ring, a subset ofcompounds having hydroxyls on the B-ring with various combinations with—OH at 3, 5, 7, positions of the A-C ring were tested.

1. Myricetin, Robinetin and Tricetin

MYR (3,5,7,3′,4′,5′-hexahydroxyflavone) and ROB(3,7,3′,4′,5′-pentahydroxyflavone) showed equivalent levels ofcardioprotection with an EC50 about 10-20 μM and TOPOIIβ and TOPOIIαinhibition at less than 10 μM. Similarly, tricetin(5,7,3′,4′,5′-pentahydroxyflavone) lacking —OH at position 3 alsodisplayed cardioprotection and TOPOII inhibition with a low level ofcytotoxicity at 100 μM. Further, 7,3′,4′,5′-tetrahydroxyflavone lacking—OH at both positions 3 and 5 of the A/C ring system displayedcardioprotection, but did not inhibit TOPOII enzymes.

However, 3′,4′,5′-trihydroxyflavone and other compounds having no —OH atthe position 7 failed to display potency less than 30. M forcardioprotection or TOPOII inhibition. These data suggested thathydroxyl (—OH) at position 7 of the A-ring may be required forcardioprotection, but not sufficient as at least one —OH group at 3and/or 5 position can greatly enhance activity (e.g., potency and/orTOPOII inhibition) of these compounds for cardioprotection. Thus,hydroxyls in the A/C-ring (3,7) system play an important role forcardioprotection and TOPOII inhibition, provided that 3′,4′,5′ hydroxylsare present on B-ring. Particularly —OH at position 7 in the A-ringappears to be critical for the activity.

Example 14. Protective Effects of MYR on Anticancer Agents

1. Anthracyclines

MYR Protects Against Epirubicin-Induced Cell Death andIdarubicin-Induced Cell Death

Epirubicin and idarubicin are anthracyclines that are associated withheart failure in patients. In addition to doxorubicin described above,the effect of MYR was tested on epirubicin- and idarubicin-induced heartinjury. As illustrated in FIG. 19 , human iPSC-derived cardiomyocyteswere mock-treated (triangle) or treated with 100 μM of MYR (circle) andincreasing concentrations of epirubicin or idarubicin for 72 hours, andthen incubated with dyes that indicate mitochondrial health and cellularnuclei as describe above. Cells were imaged and total number of healthycells were counted and plotted as percentage of mock-treatment control.

Lethal concentration at which 50% of cells were killed by epirubicin(LC50) was shifted from 0.49 μM in mock-treated to 1.28 μM inMYR-treated conditions, showing that MYR effectively protected againstepirubicin-induced cell death in cardiomyocytes (FIG. 19 , left). (n=3)

Similarly, LC50 of Idarubicin was shifted from 0.59 μM in mock-treatedto 1.04 μM in MYR-treated conditions, indicating that MYR also protectedagainst idarubicin-induced cell death (FIG. 19 , right). (n=3)

2. Protein Kinase and Proteasome Inhibitor

MYR Protects Against Bortezomib-, Sunitinib- and Sorafenib-Induced CellDeath

Cardiotoxicity may result from the formation of toxic reactive oxygenspecies (ROS) through redox cycling caused by various anticancer agent.The reactive oxygen species (ROS) may activate apoptotic pathways,leading to cell death in both cancer and normal cells. Cardiomyocytesmay be particularly sensitive to the oxidative stress and cardiacmitochondria may be easily disrupted by cancer agents likeanthracycline, TK1 or proteasome inhibitors. According to the datapresented above, it was hypothesized that the ability of MYR and itsanalogs described herein to protect heart cells can be multifaceted: (1)protection by interacting TOPOII enzymes in the heart cells as inanthracyclines; and (2) the effect exerted independently from themolecular mechanism of TOPOII (e.g., ROS chelation, promotingmitochondrial integrity). To determine whether MYR conferscardioprotection on non-anthracycline drugs, the compound was tested forits ability to protect heart cells against protein kinaseinhibitor-induced cytotoxicity.

Sunitinib and sorafenib are tyrosine kinase antagonists used to treat awide range of cancers including leukemia and sarcoma. However, sunitiniband sorafenib have been reported to cause adverse events like heartfailure in patients. Tyrosine kinases are enzymes responsible for theactivation of many proteins involved in signal transduction pathways.These proteins are activated via phosphorylation, a step the TKIs areknown to target for inhibition.

Bortezomib is a proteasome inhibitor used to treat multiple myeloma andlymphoma. In some cancer, the proteins that normally destroy cancercells are broken down prematurely. Bortezomib interrupts this process,allowing those proteins to disrupt the dividing cancer cells.

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media day 3, before performingexperiments. Human stem cell derived cardiomyocytes were then treatedDMSO, sunitinib (10 μM) or sunitinib plus increasing concentrations ofMYR (1 to 100 μM) for 72 hours, and then incubated with dyes thatindicate mitochondrial health and cellular nuclei. Cells were imaged andtotal number of healthy cells were counted and plotted as percentage ofsunitinib-treatment control. MYR displayed protection againstsunitinib-induced cell death in cardiomyocytes (FIG. 20 ). (n=3).Similarly, MYR successfully corrected more than 80% of cardiacdysfunction in 5 μM sorafenib treated cardiomyocytes (FIG. 21 ).Treatment with 100 μM myricetin also rescued bortezomib-inducedcardiotoxicity (FIG. 22 ). These data suggest that MYR protects againstprotein kinase inhibitor-induced cardiomyocyte cell death.

Example 15. No Interference with Doxorubicin's Anti-Cancer Activity

Bisdioxopiperazine dexrazoxane (DEX) is the only drug available forreducing the incidence of heart failure in cancer patients receivinganticancer agents. Despite its clinical effect, DEX is associated withseveral side effects such as interfering with antitumor efficacy ofanthracyclines, inducing secondary malignancies, and causing blood andbone marrow disorders. These limitations severely limit its use forcertain cancer patients.

The effect of MYR was investigated to determine whether the compound hasthe similar shortcomings to those observed in DEX. Breast cancer cells(MDA-MB-231) were mock-treated or treated with 100 μM of MYR and withincreasing concentrations of doxorubicin for 72 hours (FIG. 23 ). Cellviability assay was conducted (CellTiter-Glo, Promega). Luminescence wasrecorded via Synergy HT (Biotek) microplate reader and plotted aspercentage of mock-treated control. Essentially no difference wasobserved in cell viability (LC50) between mock-treated (0.53 μM) versusMYR-treated (0.48 μM), indicating that MYR does not interfere withdoxorubicin's anti-cancer activity (FIG. 23 ) (n=3).

Example 16. In Vivo Validation of Cardioprotection Against DOX-InducedToxicity

An acute anthracycline-induced cardiotoxicity model was established in9-10 week old C57BL/16 mice obtained from The Jackson Laboratory.Animals were divided into three groups; saline treated (n=8),Doxorubicin treated (n=16) or Doxorubicin+MYR treated (n=17).Doxorubicin (20 mg/kg), MYR (40 mg/kg) and saline were administered viaa single intraperitoneal injection. MYR was administered 30 minutesprior to doxorubicin treatment. General health of the animals wasmonitored on a daily basis throughout the course of the study. Mice wereanesthetized using isoflurane (˜1.0%) and transthoracic echocardiographywas performed using the VevoLAZR Imaging system (VisualSonics Inc.,Toronto, Canada) at day −4 to obtain baseline measurements and then atday 5 following the treatments. Left ventricular (LV) M-mode images wereobtained in the two-dimensional short axis view close to the papillarymuscles. Tracings of endocardial tissue during systole and diastole weremade off line. These data were then used to calculate fractionalshortening (FS) and ejection fraction (EF) which are global indices ofsystolic function.

Contractile properties were unaltered in the saline group during thecourse of the study. In contrast, doxorubicin treatment had a profoundimpact on contractile properties. In this group, FS and EF decreasedsignificantly with time (P<0.001) by 15% and 19%, respectively. MYRtreatment significantly reduced the doxorubicin-induced cardiotoxicity(P<0.05) as observed by improvement of FS and EF by 7% and 10%respectively (FIG. 24 ). At 2-fold higher concentration thandoxorubicin, MYR elicited 52% rescue of FS and 49% rescue of EFdysfunction caused by doxorubicin (FIG. 24 ).

Example 17. Effect of Various Protectants (Including Vitexin) onDoxorubicin-Induced Cardiotoxicity Regarding Mitochondrial Toxicity

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at day 3, before performingexperiments. Samples were either mock treated, treated with 1 μM ofdoxorubicin, or treated with 1 μM of doxorubicin and the indicated drugfor 48 hours. Following treatment, the samples were incubated with atetramethylrhodamine methyl ester (TMRM) dye to indicate mitochondrialhealth, and a second dye to identify cell nuclei. Cells were imagedusing the INCell Analyzer2200, and images were analyzed by CellProfilerto quantify the percentage of TMRM-negative cells. Representative dataare presented for protective agents from two independent sets of screenswhere each data point was obtained from three biological replicates.Data normalization was performed by re-calibrating data based on themock-treated sample (0% mitochondrial toxicity) and the 1 μMdoxorubicin-treated sample (100% mitochondrial toxicity).

Cardiomyocytes were either mock-treated (‘No treat’), treated with 1 μMdoxorubicin (‘Dox 1 μM’), or treated with 1 μM doxorubicin and theindicated drug, and subsequently stained to detect mitochondrial health(FIG. 25 ). Cardiomyocytes exposed to 17 μM kaempferol (‘KAE 17 μM’)exhibited a decrease in mitochondrial toxicity of at least 60%, ascompared to cardiomyocytes treated with doxorubicin in the absence of aprotective agent (‘Dox 1 μM’). Cardiomyocytes exposed to either 0.76 μMambroxol (‘AMB 0.76 μM’), 10 μM mesalamine (‘MES 10 μM’), or 50 μMN-acetyl cysteine (‘NAC 50 μM’) exhibited a decrease in mitochondrialtoxicity of at least 40%, as compared to cardiomyocytes treated withdoxorubicin in the absence of a protective agent (‘Dox 1 μM’).Cardiomyocytes exposed to either 160 μM dexrazoxane (‘Dex 160 μM’) or115 μM vitexin (‘VIT 115 μM’) exhibited a decrease in mitochondrialtoxicity of at least 30%, as compared to cardiomyocytes treated withdoxorubicin in the absence of a protective agent (‘Dox 1 M’).

Example 18. Effect of Various Protectants (Including Vitexin) onDoxorubicin-Induced Cardiotoxicity (Apoptosis)

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media day 3, before performingexperiments. Samples were either mock-treated, treated with 1 μM ofdoxorubicin, or treated with 1 μM of doxorubicin and the indicated drugfor 48 hours. Following treatment, the samples were incubated with aTUNEL dye to indicate apoptosis-positive cells, and a second dye toidentify cell nuclei. Cells were imaged using the INCell Analyzer2200,and images were analyzed by CellProfiler to quantify the percentage ofapoptosis-positive cells. Representative data are presented forprotective agents from two independent sets of screens where each datapoint was obtained from three biological replicates. Data normalizationwas performed by re-calibrating data based on the mock-treated sample(0% apoptosis) and the 1 micromolar doxorubicin-treated sample (100%apoptosis).

Cardiomyocytes were either mock treated (‘No treat’), treated with 1 μMdoxorubicin (‘Dox 1 μM’), or co-treated with 1 μM doxorubicin and theindicated drug, and subsequently stained to detect apoptosis (FIG. 26 ).Cardionyocytes treated with 115 μM vitexin (‘VIT 115 μM’) exhibited adecrease in apoptosis of at least 60%, as compared to cardiomyocytestreated with doxorubicin in the absence of a protective agent (‘Dox 1μM’). Cardiomyocytes exposed to either 160 μM dexrazoxane (‘Dex 160μM’), 0.76 μM ambroxol (‘AMB 0.76 μM’), or 50 μM N-acetyl cysteine (‘NAC50 μM’) exhibited a decrease in apoptosis of at least 50%, as comparedto cardiomyocytes treated with doxorubicin in the absence of aprotective agent (‘Dox 1 μM’). Cardiomyocytes exposed to either 17 μMkaempferol (‘KAE 17 μM’) or 10 μM mesalamine (‘MES 10 μM’) exhibited adecrease in apoptosis of at least 40%, as compared to cardiomyocytestreated with doxorubicin in the absence of a protective agent (‘Dox 1μM’).

Example 19. Vitexin Provides Long-Term Cardioprotection (MitochondrialHealth)

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at day 3, before performingexperiments. Samples were either mock-treated (FIG. 27A), treated with 1μM of doxorubicin (FIG. 27B), co-treated with 1 μM of doxorubicin and 16μM dexrazoxane (FIG. 27C), or co-treated with 1 μM of doxorubicin and116 μM dexrazoxane (FIG. 27D) for 7 days. Following treatment, thesamples were incubated with a tetramethylrhodamine methyl ester (TMRM)dye to indicate mitochondrial health. Cells were imaged using the INCellAnalyzer2200, and images were analyzed by CellProfiler to quantify thepercentage of TMRM-negative cells. Representative images are presentedfor each sample, wherein loss of TMRM signal represents mitochondrialtoxicity.

Cardiomyocytes exposed to either doxorubicin (FIG. 27B) or co-treatedwith doxorubicin and dexrazoxane (FIG. 27C) exhibited an increase inmitochondrial toxicity as indicated by a noticeable decrease inTMRM-positive cells as compared to mock-treated cardiomyocytes (FIG.27A). Treatment of cardiomyocytes with doxorubicin and vitexin (FIG.27D) demonstrated improved long term mitochondrial protection, ascompared to cardiomyocytes exposed to either doxorubicin (FIG. 27B) ordoxorubicin and dexrazoxane (FIG. 27C).

Example 20. Vitexin Provides Dose-Dependent Cardioprotection(Electrophysiological Activity)

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at day 3, before performingexperiments. Samples were either mock-treated with 0.1% DMSO, treatedwith 1 μM doxorubicin, or co-treated with 1 μM doxorubicin and variousconcentrations of vitexin (e.g., 11.6 μM, 37 μM, or 116 μM). Followingtreatment, the percentage of active electrodes in each sample wasmeasured for 72 hours. Percentage of active electrodes was quantifiedand graphed in a time course (FIG. 28A). The average number of activeelectrodes per well was quantified and graphed at 30 hours after thetreatment (FIG. 28B, n=6, standard deviation is shown as error bars).

Cardiomyocytes exposed to 1 μM doxorubicin, in the absence of vitexin,exhibited about a 50% decrease in the number of active electrodes 24hours after drug treatment (time zero), and about a 95% decrease,relative to time zero, in the number of active electrodes 30 hours afterdrug treatment (FIG. 28A). Cardiomyocytes co-exposed to doxorubicin andvitexin exhibited a dose-dependent increase in the percentage of activeelectrodes (FIG. 28A). At 24 hours following drug treatment,cardiomyocytes co-exposed to 1 μM doxorubicin and either 11.6 μM, 37 μM,or 116 μM vitexin exhibited about a 50%, about a 25%, or about a 0%decrease in the number of active electrodes, respectively. At 30 hoursfollowing treatment, samples that were co-exposed to 1 μM doxorubicinand 116 μM vitexin exhibited had a statistically significant higheraverage number of active electrodes (about 10 active electrodes) ascompared to samples that were exposed to 1 μM doxorubicin in the absenceof vitexin (about 2 active electrodes) (FIG. 28B).

Example 21. Protectants do not Inhibit Doxorubicin-Mediated Death ofBreast Cancer Cells

MDA-MB-231 cells (metastatic breast cancer) were cultured for 1 daybefore performing experiments. Samples treated with either increasingconcentrations of Doxorubicin (e.g., 0 μM, 0.016 μM, 0.05 μM, 0.16 μM,0.5 μM, 1.6 μM, 5 μM, 16 μM, or 50 μM), or co-treated with increasingconcentrations of doxorubicin and the indicated protective agent for 72hours. Cells were subsequently lysed with CellTiter-Glo reagent toidentify metabolically active (e.g., viable) cells, wherein theluminescence measured from the lysed cell suspension is directlyproportional to the number of viable cells present in the culture.Percentage cell death was quantified by measuring the decrease inluminescence. XLFit was used for curve fitting. Averages from triplicateare graphed and standard deviation is shown as error bars.

MDA-MB-231 cells co-treated with increasing concentrations ofdoxorubicin and either dexrazoxane, ambroxol, kaempferol (FIG. 29A),mesalamine, N-acetyl cysteine, or vitexin (FIG. 29B) showed nosignificant difference in the percentage of cell death as compared tocells that were treated with doxorubicin in the absence of a protectiveagent. These results indicate that the pharmaceutical compositionsdescribed herein do not confer a protective benefit to MDA-MB-231 breastcancer cells, as measured by the in vitro assay.

Example 22. Protectants do not Inhibit Doxorubicin-Mediated Death ofLung Cancer Cells

A549 cells (lung cancer) were cultured for 1 day before performingexperiments. Samples treated with either increasing concentrations ofDoxorubicin (e.g., 0 μM, 0.016 μM, 0.05 μM, 0.16 μM, 0.5 μM, 1.6 μM, 5μM, 16 μM, or 50 μM), or co-treated with increasing concentrations ofdoxorubicin and the indicated drug for 72 hours. Cells were subsequentlylysed with CellTiter-Glo reagent to identify metabolically active (e.g.,viable) cells, wherein the luminescence measured from the lysed cellsuspension is directly proportional to the number of viable cellspresent in the culture. Percentage cell death was quantified bymeasuring the decrease in luminescence. XLFit was used for curvefitting. Averages from triplicate are graphed and standard deviation isshown as error bars.

A549 cells co-treated with increasing concentrations of doxorubicin andeither dexrazoxane, ambroxol, kaempferol, mesalamine, N-acetyl cysteine,or vitexin showed no significant difference in the percentage of celldeath as compared to cells that that were treated with doxorubicin inthe absence of a protective agent. These results indicate that thepharmaceutical compositions described herein do not confer a protectivebenefit to A549 lung cancer cells, as measured by the in vitro assay.

Example 23. Acute Toxicity of Various Protectants (Including Vitexin) onElectrophysiology

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at 3 day, before performingexperiments. Samples were either mock-treated with 0.1% DMSO, or treatedwith increasing concentrations of the indicated drug for at least 20minutes. Cardiomyocytes were treated with the hERG potassium channelblocker E4031 as a control. Following treatment, the beat period andfield potential duration (FPD) were measured in each sample using theMEA.

At lower drug concentration, Cardiomyocytes exposed to eitherdexrazoxane, ambroxol, chenodeoxycholic acid, deferoxamine, N-acetylcysteine, naringenin, or vitexin exhibited no appreciable difference inbeat period or field potential duration, as compared to control samples.At higher concentrations, cardiomyocytes exposed to eitherchenodeoxycholic acid or naringenin exhibited beating cessation fromacute drug toxicity.

Example 24. Long Term Toxicity of Various Protectants (IncludingVitexin) on Electrophysiology

Cell samples were prepared by differentiating induced pluripotent stemcells into cardiomyocytes. Cells were cultured for 4 dayspost-differentiation, changing media at day 3, before performingexperiments. Samples were either mock-treated with 0.1% DMSO, or treatedwith various concentrations of the indicated drug. Following treatment,the percentage of active electrodes in each sample was measured for atleast 5 days. Percentage of active electrodes was quantified and graphedin a time course.

Cardiomyocytes exposed to either ambroxol, kaempferol, mesalamine, orvitexin showed no observable decrease in the number of active electrodesrelative to the mock-treated sample. Cardiomyocytes exposed to theclinically-approved cardioprotectant dexrazoxane exhibited a long-term,dose-dependent cardiotoxic effect. Cardiomyocytes exposed to either 167μM or 500 μM dexrazoxane exhibited about a 25% or 50% reduction in thenumber of active electrodes at about 2 days post-treatment,respectively. At about 3 days post-treatment, cardiomyocytes exposed toeither 167 μM or 500 μM dexrazoxane exhibited about a 50% or 100%reduction in the number of active electrodes, respectively.

Example 25. Treatment of Breast Cancer in a Patient with Heart Diseaseby Oral Administration of a Pill Containing Doxorubicin and Vitexin

A patient, with a history of heart disease, is diagnosed with breastcancer. Due to an increased risk for heart failure, the patient isunable to receive the standard treatment regimen of doxorubicin, whichis known to induce cardiotoxicity. Instead, the caregiver administers aco-formulation of doxorubicin (10 mg) and vitexin (100 mg). Anechocardiogram is performed and blood flow rate is measured to determineif the therapy has a cardiotoxic effect in the patient. The patientshows no indication of cardiac dysfunction. Exhibiting no signs ofcardiotoxicity, the patient is able accept higher doses of treatmentover the next several weeks. The patient subsequently undergoes a tissuebiopsy which shows no indication of breast cancer.

Example 26. Treatment of Liver Cancer in a Patient by IntravenousAdministration of Doxorubicin, Dexrazoxane and Vitexin

A patient is diagnosed with liver cancer. The caregiver administers tothe patient a co-formulation of doxorubicin (5 mg/mL) and dexrazoxane(50 mg/mL). An electrocardiogram is performed to determine if thedexrazoxane is successfully mitigating cardiotoxic effects in thepatient. The patient presents with a 20 ms QT prolongation. To enhancethe activity of dexrazoxane, the caregiver administers to the patient aco-formulation of doxorubicin (5 mg/mL) and vitexin (100 mg/mL).Following treatment, an electrocardiogram is performed, and the patientexhibits no signs of QT prolongation. The patient is able to continuereceiving treatment over several weeks after which a tissue biopsy isperformed to confirm the liver cancer has been eradicated.

Example 27. Treatment of Lung Cancer in a Patient with Bradycardia byOral Administration of a Pill Containing Doxorubicin and Myricetin

A patient is diagnosed with stage H lung cancer, and presents withbradycardia. Due to an increased risk for heart failure, the patient isunable to receive the standard treatment regimen of doxorubicin, whichis known to affect cardiac contraction and induce bradycardia. Instead,the caregiver administers a co-formulation of doxorubicin (10 mg) andmyricetin (100 mg). An electrocardiogram is used to monitor thepatient's heart rate. The patient shows no indication of cardiacdysfunction. Exhibiting no signs of cardiotoxicity, the patient is ableaccept higher doses of treatment over the next several weeks. The lungcancer is down-staged to stage 1, and the cancer is successfully removedwith surgery. Upon follow-up, a tissue biopsy is performed and shows nosign of cancer.

Example 28. Treatment of Liver Cancer in a Patient by IntravenousAdministration of a Solution Containing Doxorubicin, Dexrazoxane andMyricetin

A patient is diagnosed with liver cancer. The caregiver administers tothe patient a co-formulation of doxorubicin (5 mg/mL) and dexrazoxane(50 mg/mL). An electrocardiogram is performed to determine if thedexrazoxane is successfully mitigating cardiotoxic effects in thepatient. The patient presents with a 20 ms QT prolongation. To enhancethe activity of dexrazoxane, the caregiver administers to the patient aco-formulation of doxorubicin (5 mg/mL) and myricetrin (50 mg/mL).Following treatment, an electrocardiogram is performed, and the patientexhibits no signs of QT prolongation. The patient is able to continuereceiving treatment over several weeks after which a tissue biopsy isperformed to confirm the liver cancer has been eradicated.

Example 29. Treatment of Lung Cancer in a Patient with Bradycardia byOral Administration of a Pill Containing Myricetin

A patient is diagnosed with stage II lung cancer, and presents withbradycardia. Due to an increased risk for heart failure, the patient isunable to receive the standard treatment regimen of doxorubicin, whichis known to affect cardiac contraction and induce bradycardia. Instead,the caregiver administers myricetin (100 mg) 24 hours beforeadministration of doxorubicin (10 mg). An electrocardiogram is used tomonitor the patient's heart rate. The patient shows no indication ofcardiac dysfunction. Exhibiting no signs of cardiotoxicity, the patientis able accept higher doses of treatment over the next several weeks.The lung cancer is down-staged to stage 1, and the cancer issuccessfully removed with surgery. Upon follow-up, a tissue biopsy isperformed and shows no sign of cancer.

Example 30. Treatment of Liver Cancer in a Patient by IntravenousAdministration of a Solution Containing Doxorubicin, Dexrazoxane andMyricetin

A patient is diagnosed with liver cancer. The caregiver administers tothe patient a co-formulation of doxorubicin (5 mg/mL) and dexrazoxane(50 mg/mL). An electrocardiogram is performed to determine if thedexrazoxane is successfully mitigating cardiotoxic effects in thepatient. The patient presents with a 20 ms QT prolongation. To enhancethe activity of dexrazoxane, the caregiver administers to the patient amyricetin (100 mg) 24 hours prior to administration of doxorubicin (5mg/mL) and (100 mg/mL) intravenously. Following treatment, anelectrocardiogram is performed, and the patient exhibits no signs of QTprolongation. The patient is able to continue receiving treatment overseveral weeks after which a tissue biopsy is performed to confirm theliver cancer has been eradicated.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Such modifications areintended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporatedherein by reference in their entireties and for all purposes to the sameextent as if each individual publication or patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes.

What is claimed is:
 1. A method for preventing, reducing, or eliminatingcardiotoxicity induced by an anticancer agent in a subject, the methodcomprising administering to the subject an effective amount of aprotective agent prior to or simultaneously with the anticancer agent,wherein the protective agent is myricetin or salt thereof, wherein theanticancer agent is a protein kinase inhibitor or a proteasomeinhibitor, and wherein the administration of the protective agentprovides an effective amount of myricetin or salt thereof in thesubject, thereby preventing, reducing, or eliminating the cardiotoxicityinduced by the anticancer agent.
 2. The method of claim 1, wherein theprotective agent is administered to the subject at least about 5 minutesbefore the administration of the anticancer agent.
 3. The method ofclaim 1, wherein the protective agent and the anticancer agent areadministered simultaneously.
 4. The method of claim 3, wherein theprotective agent and the anticancer agent are co-formulated in a liquidcomposition.
 5. The method of claim 1, wherein the cardiotoxicitycomprises one or more of cardiac tissue damage, electrophysiologicaldysfunction, contractile dysfunction, DNA double strand break incardiomyocytes, mitochondrial toxicity, apoptosis, and oxidative stress.6. A method for treating cancer in a subject, the method comprising: (i)administering to the subject an effective amount of an anticancer agent;and (ii) administering to the subject an effective amount of aprotective agent prior to or simultaneously with the anticancer agent;wherein the protective agent is myricetin or salt thereof; wherein theanticancer agent is a protein kinase inhibitor or a proteasomeinhibitor; and wherein the administration of the protective agentprevents, reduces, or eliminates cardiotoxicity induced by theanticancer agent in the subject.
 7. The method of claim 6, wherein theprotective agent is administered to the subject at least about 5 minutesbefore the administration of the anticancer agent.
 8. The method ofclaim 6, wherein the protective agent and the anticancer agent areadministered simultaneously.
 9. The method of claim 1, wherein theprotein kinase inhibitor is selected from the group consisting ofafatinib, axitinib, bosutinib, cabozantinib, carfilzomib, ceritinib,cobimetanib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus,gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib,nintedanib, osimertinib, palbociclib, pazopanib, pegaptanib, ponatinib,regorafenib, ruxolitinib, sirolimus, sorafenib, sunitinib, tofacitinib,tofacitinib, temsirolimus, trametinib, vandetanib, vemurafenib, andvismodegib.
 10. The method of claim 1, wherein the protein kinaseinhibitor is a tyrosine kinase inhibitor.
 11. The method of claim 10,wherein the tyrosine kinase inhibitor is selected from the groupconsisting of sorafenib, sunitinib, bosutinib, gefitinib, dasatinib,dabrafenib, vemurafenib, imatinib, lapatinib, and nilotinib.
 12. Themethod of claim 10, wherein the tyrosine kinase inhibitor is sorafenibor sunitinib.